BBCCT-111 MEMBRANE BIOLOGY AND BIOENERGETICS

BBCCT-111MEMBRANE BIOLOGY AND

BIOENERGETICSIndira Gandhi NationalOpen UniversitySchool of Sciences

BIOENERGETICS

UNIT 7Introduction to Bioenergetics 103

UNIT 8ATP and High Energy Compounds 134

UNIT 9Oxidative Phosphorylation 148

Block

3

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BLOCK 3: BIOENERGETICSEvery living cell is a dynamic structure and has the potential to derive energyfrom its environment; convert it into a biologically useful form, and to utilize it forosmotic, chemical and mechanical work needed for the sustenance of life.Much of the molecular machinery in any cell is engaged in production andutilization of energy needed for the survival and maintains life. This Block onBioenergetics focuses on the study of the flow and transformation of energy inbiological system. Unit 7 deals with general science of energy transformationsi.e. thermodynamics and the driving force for processes. Universal electroncarriers in terms of their role and significance in harnessing and utilization ofenergy by biological systems are also dealt with. Unit 8 explains various highenergy compounds while Unit 9 on Oxidative Phosphorylation describes mito-chondrial electron transport and the mechanism how passage of electrons inthe respiratory chain creates a proton gradient across the inner mitochondrialmembrane, and the energy of this gradient is utilized to drive ATP synthesis.

Expected Learning Outcomes

After studying this block, you should be able to:

State the importance of bioenergetics and outline the significance ofvarious thermodynamic functions;

Define spontaneity and state the criterion for spontaneity of biochemicalreactions;

Discuss the role of electron carriers in energy transduction in biologicalsystems;

Understand the organization of components of respiratory electrontransport chain into complexes; and

Explain the mechanism of oxidative phosphorylation.

We hope that after studying this block you will acquire understanding of theconcepts of energetics and biosynthesis of high energy molecules.

Wishing you success in this endeavour !!

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Introduction to Biomembranes

Structure

UNIT7

INTRODUCTION TOBIOENERGETICS

7.1 Introduction


7.2 Thermodynamics and BiologicalSystems

Basic Terminology of Thermodynamics

Laws of Thermodynamics and TheirSignificance

7.3 Gibbs Energy Change

Gibbs Energy Change and Spontaneity

Gibbs Energy Change and EquilibriumConstant

Biochemical Standard State

Coupled Reactions

7.4 Non-equilibrium Thermodynamics

7.1 INTRODUCTIONAll living organisms capture, transform, store and use energy to stay alive i.e.,every living cell has the potential to derive energy from its environment; convert itinto a biologically useful form, and to utilize it for osmotic, chemical andmechanical work needed for the sustenance of life. The study of the flow andtransformation of energy in biological systems is called bioenergetics andforms the subject of this and the next few units. This, in fact is a part of a biggerdomain of study called ‘energetics’ which refers to the study of energy in terms

7.5 Redox reactions

Redox Reactions as TwoHalf-reactions

Redox Potential

Nernst Equation

Standard TransformedReduction Potential, E’°

Gibbs Energy and ReductionPotential

7.6 Universal ElectronCarriers

7.7 Summary

7.8 Terminal Questions

7.9 Answers

7.10 Suggested Readings

“In every one of us there is a living process of combustion going on very similar to thatof a candle, and I must try to make that plain to you.”

—Michael Faraday, 1860

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of its flow and transformation within a system or between a system and itssurroundings. In simpler words, we can say that bioenergetics deals with thegeneration of energy by harnessing the chemical energy of nutrients or lightenergy from solar radiation (in phototrophs) and storage & utilization of energyfor performing different cellular functions. It also helps us understand myriads ofbiochemical reactions interconnected through complex pathways in the cell.

We would begin the unit by recalling the basic terminology and the laws ofequilibrium thermodynamics about which you would have learnt in your earlierclasses. In this context, we would also recall thermodynamic functions viz.,energy, enthalpy, and entropy and discuss their significance. Gibbs energy isprobably the most important concept required to understand bioenergetics. Wewould give a detailed account of Gibbs energy, its importance as a criterion forspontaneity, and its relationship with equilibrium constant. Thereafter, wewould discuss, in brief, about the need and relevance of ‘non-equilibriumthermodynamics’ in the study of biological systems.

Further, as one of the important means of energy transfers in the livingsystems is through redox reactions, so we would take up the basic aspects ofredox reactions. Herein we would talk about redox reactions, redox potential,standard reduction potential and their significance. We would also introducethe need for biochemical standard state, define it and discuss, how do theparameters like Gibbs energy and redox potential change under biochemicalstandard state and what is its significance. Towards the end of the unit, wewould take up universal electron carriers in terms of their role and significancein harnessing and utilization of energy by biological systems. In the next unityou would learn about ATP and other high energy compounds.

Expected Learning OutcomesAfter studying this unit, you should be able to:

define bioenergetics and state its importance;

define and outline the significance of various thermodynamic functions;

state and explain the First and Second Laws of thermodynamics;

define spontaneity and state the criterion for spontaneity of biochemicalreactions;

define Gibbs energy and outline its significance;

relate Gibbs energy to the equilibrium constant;

state the need for biochemical standard state and define it;

explain coupling reactions and discuss their significance;

outline the need for non-equilibrium thermodynamics;

define oxidation and reduction;

explain the terms like standard reduction potential, cell potential and redoxpotential and state their significance;

relate Gibbs energy with standard reduction potential; and

discuss the role of electron carriers in energy transductionin biologicalsystems.

Block 3 Bioenergetics

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7.2 THERMODYNAMICS AND BIOLOGICALSYSTEMS

Thermodynamics is an experimental science based on certain generalisationsformulated on the basis of extensive observations of the universe we live in.These generalisations are expressed in terms of laws of thermodynamics thatgovern all physical, chemical, and biological processes. In order to understandthese laws and their application to biological systems, it is pertinent to recallthe basic terminology of thermodynamics.

7.2.1 Basic Terminology of ThermodynamicsLet us recall the basic terminology of thermodynamics about which you wouldhave learnt in your earlier classes.

System and its types: A ‘system’ in thermodynamics is defined as that part ofthe universe, which is under consideration for the study. The system can belarge or small, simple or complex e.g., a single cell or the whole organism.Further, a thermodynamic system must have well defined boundary thatseparates it from the surroundings. The surroundingsin turn,are theremaining (other than the system) part of the universe. The system and itssurroundings put together constitute the universe. However, for practicalpurposes, the surroundings can be taken as that part of the universe withwhich the system can interact.

If the boundary of the system is such that it allows the transfer of matter andenergy between the system and its surroundings through it, the system isclassified as open system. A glass test tube containing hot water is anexample of an open system as water can be removed or more water or someother kind of matter can be added to it and also the temperature of water canbe increased (by heating it) or decreased (by allowing it to cool down) bysuitable transfer of energy. A living cell is also an example of an open systemas there is a continuous flux of energy and matter to and from it.

All living organisms areopen systems as theydo exchange matter(nutrients and wasteproducts) and energy(as heat) with theirsurroundings

If the boundary of the system does not permit the transfer of matter between thesystem and its surroundings, but allows transfer of energy, it is called a closedsystem. If the glass test tube containing hot water were suitably capped suchthat the matter exchange is not allowed, it would become a closed system.Further, a system having a boundary that does not permit the transfer of eitherheat or matter to or from the surroundings is termed as an isolated system. Ifthe suitably capped glass test tube containing hot water is wrapped with a thickthermally insulating material, it will become an isolated system. Different typesof thermodynamic systems are schematically shown in Fig.7.1.

Unit 7 Introduction to Bioenergetics

Fig. 7.1: Schematic representation of different types of thermodynamic systems

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State of a system: At any instant the system is in a thermodynamic state thatcan be defined by specifying the thermodynamic properties like, pressure, p;volume, V; temperature, T; amount, n; and density, , etc. of the system. Forexample, 50 cm3 of distilled water at 30oC in a beaker constitutes a state (thepressure here will be 1 atm.). If we change any of these variables of thesystem we get a new state. For example, if we heat the water in the beaker to70oC, we would get to a new state of the system. Further, the act of changingthe state of a system is called a thermodynamic process. In this example ofheating the water in a beaker, we have performed a thermodynamic process.

7.2.2 Laws of Thermodynamics and TheirSignificance

As stated above, the laws of thermodynamics are generalisations that arebased on observations of the macroscopic world. These are physical laws ofnature that govern how the nature works. Contrary to the laws of society welive in, there has not been any violation of the thermodynamic laws.

The first law of thermodynamics concerns conservation of energy i.e., in anyphysical or chemical process the total energy of the universe (system +surroundings) remains constant. In other words, the energy can neither becreated nor destroyed; it can only change forms. The first law is empirical innature and cannot be derived mathematically from more fundamentalprinciples. However, to understand it we need to recall the concept of internalenergy (U) of the system, which is the energy possessed by a given systemby virtue of its very existence and includes all possible types of energies,which all the constituents of the system put together can have. The internalenergy of a system is a state function i.e., its value depends only on the stateof the system and not on how it has been achieved. Further, it is important tonote that the absolute value of the internal energy of any system cannot beknown, however the change in internal energy, U is measurable. If a systemexchanges some heat (q) with the surroundings and performs some work (w)then we can mathematically express the First Law of thermodynamics as

U = q + (-w) …(7.1)

Where, U is the change in the internal energy of the system. The living cellsare capable of interconverting different types of energies. The chemical energystored in the food we consume is converted partly to high-energy bonds in ATPand other molecules and rest is released as heat. On a wider scale, we cansee that the solar energy (as sunlight) is converted to chemical energy duringphotosynthesis, and this is utilized in the body for mechanical work (musclecontraction), chemical work (biosynthetic work) and osmotic work (movementof molecules across the membranes).

Enthalpy is a property closely related to internal energy. It is defined as

H =U + pV …(7.2)

and corresponds to the heat exchanged by the system with the surroundingsunder the conditions of constant pressure if the only work done is pressure-volume work. However, if no gases are involved in the process of interest, saya biochemical reaction then the change in volume is negligible and the

The thermodynamicvariables are also calledstate variables as thesedepend on the state ofthe system and not onthe past history of thesystem.

The first law can also bestated, as “the energy ofan isolated system isconstant”

As per the signconvention the heatgiven to the system ispositive and the heatgiven by the system isnegative. Similarly thework done on thesystem is positive andthe work done by thesystem is negative.


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enthalpy change is roughly same as the change in internal energy. That is whyin biological systems “energy” and “enthalpy” are generally usedinterchangeably. A process (or a biochemical reaction) accompanied byrelease of energy as heat to the surroundings is called an exothermicprocess; whereas the one accompanied by absorption of energy as heat fromthe surroundings is termed as an endothermic process.

The Second Law of thermodynamics concerns the spontaneity or thedirectionality of a process. A spontaneous process is the one that occurs onits own under a given set of conditions without the aid of any external agency.For example, hot water in a container spontaneously cools down to thetemperature of its surroundings. The spontaneity of a process can beascertained in terms of another thermodynamic property called entropy whichliterally means “a change within”. It is denoted as S, and is a measure ofrandomness of the system; greater the randomness higher the entropy. Anordered state (say a solid) is a low-entropy state, whereas a relativelydisordered state (say a liquid) is a high-entropy state. Thermodynamically,entropy is defined in terms of the heat change and temperature of the system.In a given reversible process (the one that occurs in an infinite number of verysmall steps) the entropy change, dS, is given as the ratio of heat produced,dq, and the temperature at which it is produced.

dS = dqrev

T …(7.3)

The entropy can also be visualized as a measure of dispersal of energy andmatter, greater the dispersal higher the entropy. On a microscopic level theentropy can be quantitatively interpreted in terms of the number of ways inwhich the molecules of a system can be arranged while keeping the totalenergy of the system constant.

S = k lnW …(7.4)

This number is called the number of microstates (W); a system with moremicrostates has greater entropy.

According to the Second Law of thermodynamics for a process to bespontaneous, the total entropy change for the system and surroundings( Ssys + Ssurr) or universe ( Stotal) must be positive (> 0) i.e.,

Stotal = Ssys + Ssurr > 0 …(7.5)

It is important to note that the entropy change for the system can be negativeor positive. If for a spontaneous process the entropy decreases within asystem, but it is accompanied by a greater increase in the entropy of thesurroundings. As a result, the total entropy change is positive and the processis spontaneous. Thus, according to the Second Law, entropy change providesa criterion for spontaneity of a process however; its direct application todifferent systems is quite limited. This is so because it requires calculation ofentropy changes for the system as well as for the surroundings which are verydifficult. Gibbs energy change provides a relatively more convenient criterionfor spontaneity that depends only on the properties of the ‘system’. Let uslearn about the Gibbs energy and its significance. However, before thatanswer the following simple questions to assess your learning.

You may note thatentropy provides adescription of energydistribution, but it is nota representation ofenergy that is availablefor useful purposes.

Sum of the entropychange of a system andits surroundings (orcombined entropy of thesystem and thesurroundings) alwaysincreases for aspontaneous process,

Stotal > 0


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G for a process refersto the amount of energythat is available toperform non-pV work.

Define the types of thermodynamic systems giving suitable examples.

What are spontaneous reactions? What is the criterion for spontaneity of areaction?

7.3 GIBBS ENERGY CHANGEGibbs energy (G), named after American scientist Josiah Willard Gibbs,is ameasure of the net driving force of a process under the conditions of constanttemperature and pressure (which is applicable to most biological systems)and is defined as

G = H–TS …(7.6)

The Gibbs energy, like enthalpy and entropy, is a state function. Its absolutevalue for a system cannot be determined. However; it is possible to determinethe change in Gibbs energy in a process. It is given as

G = H–T S …(7.7)

The enthalpy change H, for a reaction equals the difference in the enthalpiesof the products and the reactants, whereas the entropy change S gives thechange in the system’s randomness.

As you know, the enthalpy change essentially is a measure of the type andnumber of bonds formed and broken in the reaction. It equals the amount ofenergy exchanged as heat with the surroundings under conditions of constanttemperature and pressure. However, all this energy is not available for usefulwork. Of this, a part of energy equal to T”S is dispersed to increase theentropy, and is not available to do work, and the rest equal to G is the energyavailable for useful work, which could be mechanical work, synthetic(chemical) work or osmotic work. It is because of this fact that Gibbs energywas earlier referred to as ‘Gibbs free energy’ or just ‘free energy’ however; thisterm is no longer in use as lUPAC (the International Union of Pure and AppliedChemistry) has recommended the term ‘free’ to be dropped.

It may be noted that heat can do work only when it goes from an object athigher temperature to another at lower temperature. As the biochemicalreactions occur under conditions of constant temperature and pressure, thecells cannot use heat as source of energy. The cells can only use Gibbsenergy, which they harness from nutrient molecules (in heterotrophs) or fromsolar radiation (in phototrophs) and generate ATP molecules that act as theenergy currency of the cell. Gibbs energy acts as a criterion for spontaneity ofa reaction, and also provides information about the equilibrium position.

7.3.1 Gibbs Energy Change and SpontaneityLet us see how does change in Gibbs energy ( G) provide a criterion forspontaneity. You would recall that as per the Second Law of thermodynamicsthe total entropy change in a process must be positive, Eq. (7.5)

Gibbs energy change,G, is a quantitative

measure of the netdriving force of aprocess (at constanttemperature andpressure).

Biologists andbiochemists generallyuse Gibbs energy ratherthan entropy in theirthermodynamiccalculations.

Thus, the change inGibbs energy is theamount of enthalpy (orheat) that is “free” to beconverted into usefulwork; the rest is involvedin entropy change.


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Stotal = Ssys + Ssurr > 0 …(7.5)

For a closed system under the conditions of constant temperature andpressure, the enthalpy change can be equated with the heat flow across thewalls of the system to the surroundings. The surroundings are much largerthan the system, and are under conditions of constant temperature andpressure. Under such conditions, the heat transferred into the surroundings,qsur, is equal to the change in the enthalpy of the system. We can, therefore,write

Ssurr =Hsys

T …(7.8)

Substituting in Eq. (7.5), we get

…(7.9)

Multiplying the equation by –T, and simplifying we get

T Stotal = T Ssys + Hsys < 0 …(7.10)

It implies that for a spontaneous process, the -T Stotal term should benegative. As the temperature is always positive it is the same as saying thatthe Stotal must be positive (Eq.7.5). Thus, in terms of the properties of systemwe can give the criterion for spontaneity as

Hsys T Ssys < 0 …(7.11)

or Gsys < 0 …(7.12)

In other words, we can say that for a thermodynamic process to bespontaneous its Gibbs energy must decrease or the change in Gibbs energyshould be negative (i.e., G< 0). You may note here that in Eq. (7.11) theenthalpy change and entropy change, both are for the system only.

Chemical (or biochemical) reactions accompanied by a decrease in Gibbsenergy ( G < 0) are spontaneous and are called exergonic (Greek: ‘workproducing’), whereas the ones associated with an increase in the Gibbsenergy ( G > 0) are non-spontaneous and are called endergonic (Greek:‘work consuming’) reactions. Schematic Gibbs energy profiles for endergonicand exergonic reactions are given in Fig. (7.2).

Gibbs energy change( G) expresses energyexchange in terms ofspontaneity and theamount of useful energy.

It is important to knowthat though “G tellsabout the spontaneity ofa reaction but it gives noclue about the rate ofthe reaction. The timeaspect falls in thedomain of chemicalkinetics.

Fig. 7.2: Schematic Gibbs energy profiles for a) endergonic and b) exergonicreactions


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In exergonic reactions Gibbs energy becomes available for useful work,whereas in endergonic reactions, we need to provide Gibbs energy for thereaction to occur. The reactions in which G is equal to zero ( G = 0) impliesthat these are at equilibrium. In other words, in such cases the rate of forwardreaction (formation of products) is equal to the rate of backward reaction(formation of reactants) i.e., there is no net change in the concentrations of theproducts and reactants.

Further, to use G as a criterion for spontaneity we need to consider thetemperature as well as changes in enthalpy and entropy of the system. Areaction that is favorable from enthalpy point of view ( H <0) may not bespontaneous if the entropy also decreases ( S <0) to an extent such that the -T S term becomes more than the decrease in enthalpy and G becomespositive. On the other hand, an endothermic process ( H >0) can bespontaneous if it is accompanied by a large increase in entropy. In such acase -T S term becomes responsible for making G negative. The interplay of

H, S and T in deciding the spontaneity of a process is schematicallyrepresented in Fig. 7.3.

Fig. 7.3: Schematic representation of dependence of spontaneity of areactionon H, S and T.

Having learnt about Gibbs energy change and its significance as a criterion forspontaneity, answer the following simple question to assess your learning.

Define Gibbs energy and outline its significance.

7.3.2 Gibbs Energy Change and EquilibriumConstant

We have stated above that the Gibbs energy change provides informationabout the directionality of a reaction. To understand this, let us take a simple


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reaction in which a reactant A gets converted into product B and there isequilibrium between A and B, which can be represented as

A B …(7.13)

At equilibrium, the concentrations of A and B do not change with time and arerelated in terms of equilibrium constant, K

…(7.14)

The magnitude of K is a measure of the extent to which the reaction proceedsin the forward direction. A large value of Kindicates that the equilibriummixture contains primarily the product B, whereas a small value of K suggeststhe predominance of the reactant A in the reaction mixture. The ratio of theconcentrations of B to A at any instance, other than that at equilibrium, iscalled mass action ratio or reaction quotient, Q. It has the same expressionas the equilibrium constant, the only difference is that the concentrations areat any instance other than the equilibrium.

Q =[B] …(7.15)[A]

The value of reaction quotient ranges from 0 (corresponding to pure A) toinfinity (corresponding to pure B).For a general case in which ‘a’ moles ofsubstance A react with b moles of substance B to give c moles of substanceC and d moles of substance D as per the following expression:

aA + bB cC +dD …(7.16)

The expressions for the equilibrium constant and the reaction quotients wouldrespectively, be

…(7.17)

The Gibbs energy change for the reaction given in Eq. (7. 16) is given as

rG rGo +RT in Q …(7.18)

…(7.19)

Here, rGo is the standard Gibbs energy change, R and T being the

universal gas constant (8.314 J K-1mol-1) and absolute temperature in kelvin(K), respectively. The quantities within the square brackets represent theconcentrations (rather the activities, to be more precise), of the respectiveproducts and reactants. The magnitude of Gibbs energy change ( rG) is ameasure of the tendency of the system to move towards equilibrium.

You can note that rG depends on two quantities viz., rGo and RT ln Q. For a

given reaction at temperature T, the value of rGo is fixed. However, the value

of Q in the second term varies according to the composition of the reactionmixture. If the reaction is carried out under standard conditions i.e., theconcentrations (rather activities) of reactants and products being maintainedat unity (1M for solutions and one bar for gases) then the second term in

Activity is ‘effectiveconcentration’. It can beseen as theconcentration of thespecies corrected for itsnon-ideal behavior atmoderate or highconcentrations. In highlydilute solutions theactivity is same asconcentration.


Kc = [C]eq [D]eq

and Q= [C]c [D]d

[C]eq [D]eq Q= [C]c [D]d

dc

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Eq.(7.19) becomes zero (lnK= ln1=0), and

rG = rGo …(7.20)

Thus, the standard Gibbs energy change refers to the Gibbs energy changefor a reaction in which all the reactants and products are at unit activity. In otherwords it is a measure of the tendency of the system to move towards theequilibrium when the system is at standard conditions. However, the actualGibbs energy change depends on the prevailing concentrations of thereactants and products. It is important to note that the criterion for spontaneityof a reversible reaction is rG < 0 and not rG

o < 0.

As stated above, at equilibrium, rG = 0 and Q= K; so we can write,

0 = rGo + RT In K …(7.21)

rGo = - RT In K …(7.22)

This provides an important link between the thermodynamic quantities andequilibrium constant. Substituting it back in Eq. (7.18) we get,

rG = - RT In K + RT In Q …(7.23)

rG = - RT In K = -2.303RT log K …(7.24) Q Q

Thus, if Q<K, rG is negative and the reaction is spontaneous, andapproaches the equilibrium where the rG becomes zero (as K=Q). On theother hand, if Q >K, rG is positive and the reaction is spontaneous in thereverse direction, and once again approaches the equilibrium where rGbecomes zero (as K=Q).

Let us take the reaction given in Eq. 7.13 again and start with pure A; the valueof Q will be close to zero ( rG < 0), and the reaction would go in the forwarddirection, and some B will be formed. As more and more of B is formed, thevalue of Q goes on increasing and becomes lesser and lesser negative. Oncethe equilibrium is established, Q becomes equal to K and rG becomes zero.Similarly, if we start with pure B, the value of Q will be close to infinity ( rG > 0)for the reaction as written, and the reaction would go in the backward direction(as the reverse reaction will be spontaneous now) and some A will be formed.As more and more of A is formed, the value of Q goes on decreasing, and rGbecomes lesser and lesser positive. Once again when the equilibrium isestablished Q becomes equal to K and rG becomes zero.

From this discussion, we can conclude that

The Gibbs energy, G of the reaction is at its minimum at equilibrium, and itincreases as we go away from the equilibrium.

Approach to the equilibrium is spontaneous from either direction

Any fluctuation in the system at equilibrium will increase the Gibbs energy;however, the system will spontaneously relax back to the equilibrium.

The Gibbs energy content of the reaction (A B) as a function of itsdisplacement from equilibrium for endergonic and exergonic reactions isschematically shown in Fig. 7.4.

The standard Gibbsenergy change ( rG

o) canbe seen as an alternativeway of expressing theequilibrium constant forthe reaction.

It is important to notethat the equilibriumconstant is related to thestandard Gibbs energychange ( rG

o ) ratherthan to the actual Gibbsenergy change ( rG) forthe reaction.


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Fig. 7.4: Gibbs energy content for the reaction, A B as a function of itsdisplacement from equilibrium for a) exergonic reaction and b) endergonicreaction.

Now come back to the standard Gibbs energy change, rGo. It is equal to thedifference in the standard molar Gibbs energies of the products and thereactants, i.e.,

…(7.25)

The molar Gibbs energies of the reactants and products in turn can be givenin terms of their standard Gibbs energies of formation. So we can write

…(7.26)

As you know, the Gibbs energy of formation, fGo of a compound is defined asthe standard Gibbs energy change for the reaction involving formation of thecompound from its elements in their reference states, which are taken to bezero.

7.3.3 Biochemical Standard StateIn the context of defining standard Gibbs energy change we have defined thestandard state for a reaction to be the one in which all the reactants andproducts are at unit activity (or 1M concentration for convenience). If thereaction involves H+ ions, the standard state would correspond to a pH=0. Asyou know, biochemical reactions take place in the cell at a pH of about 7.0. Insuch a situation, it would not be appropriate to use the standard Gibbs energyvalues based on the standard state (as given above) which is usually appliedin physical chemistry. Therefore, there is a need to adopt a different standardstate for biochemical reactions.

In case of biochemical reactions we use a standard state in which theconcentration of all the species are taken to be 1M as before, but theconcentration of H+ ions is taken to be 1.0 x 10-7M (i.e., pH = 7.0).In addition,the concentration of H2O is taken to be constant as 55.5 M, and in thereactions involving Mg2+ ions, their concentration is assumed to be constantand taken as 1 mM = 1x10-3M, and the ionic strength, I=0.25. Further, in orderto make biochemical standard Gibbs energy to be distinct, it is given adifferent symbol, rG

o and is called standard transformed Gibbs energy

It may be noted that twodifferent symbols are inuse for standard Gibbsenergy change as perbiochemical standardstate. These are rGo’

and rG’ o. However,

rG’ o is the onerecommended byinternational committeeof chemists andbiochemists.


rGo Go (products) Go (reactants)m m

rGofGo (products) fGo (reactants)

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change. For a given reaction, the standard transformed Gibbs energy change( rG o ) is a constant, and is related to the equilibrium constant of the reactionas per the following equation.

rG o = -RTIn K …(7.27)

Here, K refers to the transformed equilibrium constant. Similarly, otherthermodynamic properties of the system based on biochemical standard stateare called standard transformed constants e.g., rH is called standardtransformed enthalpy change and so on. Let us take an example to see therelationship between standard Gibbs energy change for a system usingphysical and biochemical standard state.

Let’s consider a general reaction in which one of the products happens to be ahydrogen ion.

A + B C +H+ …(7.28)

For this reaction we can write the Gibbs energy change as

…(7.29)

Under conditions of physical standard state (all species at 1M concentration)

rG rGo …(7.30)

Now, under the conditions of biochemical standard state the expression for theGibbs energy change becomes

…(7.31)

Equating, Eq. (7.30) and Eq. (7.31), we get

…(7.32)

Substituting the values of R and T, and converting natural log to the log tothebase 10, we get

…(7.33)

Solving, we get

…(7.34)

Similarly, for a biochemical reaction involving one of the reactants to be ahydrogen ion, we can show that

…(7.35)

Similar to rGo being equal to the difference in the standard molar Gibbs

energies of the products and reactants, rGo is the difference in the standard

molar Gibbs energies of the products and reactants under biochemicalstandard conditions. As discussed above, for rG

o, in the present case rGo

gives the tendency of a system under biochemical standard state to movetowards the equilibrium. We have so far talked about three different versions ofthe Gibbs energy change for a given reaction. It is worthwhile to summarisethem to understand differences between them.

Physical constantsbased on thebiochemical standardstate are also calledstandard transformedconstants.

If a reaction does notinvolve water, hydrogenion or any ionisablespecies then thestandard Gibbs energyaccording to biochemicalstandard state is sameas that for physicalchemistry standardstate.


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rG : it is called Gibbs energy change for the reaction and equals thedifference between the Gibbs energies of the products and the reactants whenthe system is not at equilibrium. It is the driving force behind the system goingto (attaining) the equilibrium.

rGo : it is the standard Gibbs energy change of the reaction and equals theGibbs energy change of the reaction when all the reactants and products areat physical chemistry standard state [concentration of 1 M or a partialpressures of 101.3 kPa (for gases) and temperature of 298 K ]. It is the drivingforce behind the system initially at standard state going to (attaining) theequilibrium.

rG o : it is the standard transformed Gibbs energy change of the reaction andequals the Gibbs energy change of the reaction when all the reactants andproducts are in their biochemical standard state [concentration of 1 M or apartial pressures of 101.3 kPa (for gases), temperature of 298 K, [H+] =1x 10-7M; [[H2O]=55 M], [Mg2+]= 1x 10-3 M. It is the driving force behind thesystem initially at biochemical standard state going to the equilibrium.

Having learnt about biochemical standard state and the relationship betweenthe standard Gibbs energies of reaction expressed in terms of physical andbiochemical standard states respectively, let’s take up coupling reactions, oneof the important strategies used by the biological systems to transformchemical energy of the nutrients and light energy for their sustenance.

7.3.4 Coupled ReactionsYou know by now that in bioenergetics we deal with the way living cell getsenergy from fuel metabolism or light capture and how it uses it for drivingenergy-requiring reactions. You also know that cellular functionality dependson the formation and activity of molecules like proteins and nucleic acids. Theformation of these ‘ordered’ macromolecules is energy intensive i.e., the cellneeds energy ( G is positive) to synthesize them from smaller molecules.The question remains, “How do these endergonic reactions occur?”

The answer lies in coupled reactions. In such reactions thermodynamicallyunfavorable i.e., endergonic reactions are combined or coupled with energyliberating i.e., exergonic reactions, such that the Gibbs energy change foroverall reaction is negative and the process is exergonic (spontaneous). Thethermodynamic basis of coupling of endergonic and exergonic reactions isthat the two reactions share a common intermediate. That is, the product ofone reaction is a reactant in the other reaction. If, for an arbitrary case,

Reaction 1: A + B C rG’o =+5 kJ mol-1 …(7.36)

Reaction 2: C + D E rG’o =-12 kJ mol-1 …(7.37)

Net Reaction: A + B + D E rG’o =-7 kJ mol-1 …(7.38)

The Gibbs energy for the net reaction can be obtained by algebraic summationof those of the individual coupled reactions. Let us understand it with the helpof a biochemical example.

Example1: The formation of adenosine triphosphate (ATP) from adenosinediphosphate (ADP) involves a reaction of ADP with inorganic phosphate (Pi)

You may note that theG values are additive.

This in fact is aconsequence of Gibbsenergy being a statefunction, which meansthat its value for asystem depends on thestate of the system,irrespective of how ithas been achieved. Inother words, whetherthe state is obtained inas single step or in anumber of steps.

Further, the mechanismof a reaction also hasno effect on G e.g.,the G for the oxidationof glucose is the samewhether it occurs bycombustion in a testtube or by a series ofenzyme-catalyzed


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as given below, Eq. (7.39). It is an non-spontaneous reaction ( G>0) andwould not occur without an input of energy.

ADP + Pi ATP + H2O rG’o =+30.5 kJ mol-1 …(7.39)

In many of the metabolic pathways in the cell, ATP is produced by coupling thereaction between ADP and Pi to another reaction that releases enough energy(more than that required for formation of ATP) to make the coupled reactionspontaneous. In the glycolytic pathway (which you would have studied in yourearlier classes) the formation of ATP is coupled with the conversion of 1,3-bisphosphoglycerate into 3-phosphoglycerate, Eq. (7.40).

1,3-Bisphosphoglycerate + H2O

3-phosphoglycerate + Pi; rG’o= 49.4 kJ mol-1 …(7.40)

The coupling between the two reactions is brought about by the enzyme,phosphoglycerate kinase that catalyses both the reactions. The net reactioncan be obtained by combining Eq. (7.39) and Eq. (7.40) and simplifying asfollows

1,3-Bisphosphoglycerate + H2O 3-phosphoglycerate + Pi; rG’o= -49.4 kJ mol-1

ADP + Pi ATP + H2O rG’o =+30.5 kJ mol-1

1,3-Bisphosphoglycerate + ADP 3-phosphoglycerate + ATP rG’o = 18.9 kJ

You may note here that the net reaction is spontaneous ( rG’o<0; exergonic).

The overall Gibbs energy change for the coupled reaction can be used tocalculate the equilibrium constant for the reaction. We can take one moreexample of coupling reaction from glycolytic pathway to understand this.

Example 2: The very first step of glycolysis involves the conversion of glucoseto glucose-6-phosphate, again an endergonic reaction, Eq. (7.41).

Glucose+ Pi glucose- 6- phosphate; rG’o= 13.8 kJ mol-1 …(7.41)

The required Gibbs energy is provided by hydrolysis of ATP,

ATP + H2O ADP + Pi rG’o = -30.5 kJ mol-1 …(7.42)

The net reaction is

Glucose+ ATP + H2O glucose- 6- phosphate + ADP rG’o = -16.7 kJ mol-1

…(7.43)

This reaction is catalysed by the enzyme hexokinase that facilitates thetransfer of phosphate group from ATP to glucose. As you know that rG’o andthe equilibrium constant are related, we can calculate the equilibrium constantfor this reaction as follows.

rG’o = - RT InK’ …(7.27)

Rearranging, we get…(7.44)

Substituting the value of the standard transformed Gibbs energy change, Rand T (298K) we get, K´ = 846

In a coupled reaction aspontaneous reactionprovides the energyneeded by asubsequent non-spontaneous reaction.


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You may solve the following simple questions to assess your understanding oftransformed Gibbs energy change and coupled reactions.

Derive the relationship between the standard Gibbs energy changes for thefollowing reaction expressed in terms of physical and biochemical standardstates respectively.

A + nH+ B + C

What are coupled reactions? Discuss their importance giving a suitableexample.

7.4 NON-EQUILIBRIUM THERMODYNAMICSAs stated above, a living cell is an open system; there is a continuous flux ofenergy and matter into and out of the cell. At a given instance it is in a steadystate i.e., the concentrations of different constituents in it remain constant overa period of time. In other words, the input of energy (and matter) and output ofenergy (and matter) is equal. As the continuous flow of matter across thecellular boundaries does not allow the establishment of a true equilibrium, thelaws of thermodynamics (equilibrium thermodynamics to be specific) are notcompletely applicable to the cell. Though under the conditions of constanttemperature and pressure, if we focus our attention on to a set of reactions inthe cell, the system is quite close to equilibrium and the thermodynamic lawsare fairly applicable. However, a better and detailed study of cellular reactionsrequires a non-equilibrium thermodynamic treatment. This is a domain ofstudy, which was recognised more than a hundred years ago, and is still notfully established. A detailed account of non-equilibrium thermodynamics isbeyond the scope of this course.

Oxidation-reduction (redox) reactions play very crucial role in the supply andtransformation of energy in biological systems. Let us recall the basic aspectsof redox reactions and learn about their roles.

7.5 REDOX REACTIONSYou would recall from your earlier studies that the oxidation involves the lossof one or more electrons by an atom, molecule or an ion. In the followingreaction, the ferrous ion is getting oxidised to ferric ion by the loss of oneelectron.

…(7.45)

Reduction, on the other hand, is the process that involves the gain of one ormore electrons by an atom, molecule or an ion. In the following reactionchlorine molecule get reduced to chloride ion by gaining electrons

…(7.46)


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Further, you would also recall that the oxidation and reduction processesalways occur together in a reaction. In other words, the loss of electrons byone substance is always accompanied by a gain of electron by some otherspecies, and consequently there are no free electrons in a chemical system.Such reactions involving oxidation and reduction processes are termed redoxreactions or electron transfer reactions.

In a reaction, the species possessing higher affinity for electrons cause theoxidation of other substances by abstracting electrons from them. Thesespecies are called oxidising agents or oxidants. In the process, the oxidisingagents themselves get reduced (as they gain electrons). In biologicalsystems, oxidations can take place in a variety of ways, such as addition ofoxygen, or removal of electrons or removal of hydrogen (dehydrogenation). Ofthese, dehydrogenation is the most common form of biological oxidation. Onthe other hand, the species that readily give up electrons cause the reductionof other substances. These are called reducing agents or reductants, andin the process of reducing a substance they themselves get oxidised.

Work done in living systems depends on flow of electrons in oxidation-reduction reactions occurring in them. For example, in heterotrophs food actsas a source of electrons. The food molecules in the reduced form are oxidised(e.g., glucose is oxidised to carbon dioxide and water), and the electronsobtained in this process move through different metabolic intermediates todedicated electron carriers in enzyme-catalyzed reactions. In other words,these organisms consume the organic materials produced by photosyntheticorganisms and use them as metabolic fuel. The electron carriers in turn,donate these electrons to species with higher electron affinities. This isaccompanied by release of energy, which is used for performing different typesof work. You would learn about these electron carries in the next section. Onthe other hand, the phototrophs use energy from light to produce chemicalenergy in the form of nutrients like glucose which also involves redoxreactions.

7.5.1 Redox Reaction as Two Half-reactionsContinuing with the basic aspects of oxidation-reduction reactions, let us takean example of a redox reaction. You are familiar with Benedict reaction used tocheck for the presence of reducing sugars like glucose.

…(7.47)

In this reaction, the cupric ions are reduced to cuprous oxide by the aldehydegroup in glucose; which itself is oxidised to carboxylic acid group. Though bothoxidation and reduction occur simultaneously in redox reactions, it isconvenient to consider the oxidation and reduction reactions separately as twohalf-reactions while describing electron transfers. In the above case, we canwrite,

(Oxidation) …(7.48)

(Reduction) …(7.49)

The sum of the two half-reactions gives the whole reaction. This method ofexpressing a redox reaction as two half-reactions provides a kind of flexibility to

The redox reactionsinvolve the loss ofelectrons by onechemical species,which is therebyoxidized, and the gainof electrons by another,which is reduced


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the concept of redox reactions. However, you may note here that this idea oftwo half-reactions is for convenience of understanding; there are no freeelectrons in a reaction. The electrons in these conceptual reactions can beseen as being “in transit”. In general, these half-reactions can be representedas conjugate redox pairs or redox couples,

Electron donor (1) Electron acceptor (1) + e– …(7.50)

Electron donor (2) + e– Electron acceptor (2) …(7.51)

Net reaction:

Electron donor (1) + Electron acceptor (2) Electron acceptor(1) +Electron donor (2) …(7.52)

The redox couple of oxidized and reduced species in a half-reaction isdenoted as Oxidised form/Reduced form e.g., Fe3+/Fe2+. Thus, in a redoxreaction, there would be two conjugate redox pairs and there would bespontaneous electron transfer from the electron donor of one redox pair to theelectron acceptor of the other. Now, questions arise that in a redox reaction,which species would act as an oxidising agent and which would be a reducingagent? How do we determine that? Also, which of the two oxidising or reducingagents is stronger, and why? Qualitatively speaking, a species possessingstronger affinity for electrons cause the oxidation of other species byabstracting electrons from it and act as oxidising agent. On the other hand,the species that readily gives up electron act as reducing agent and cause thereduction of other substances.

Quantitatively, the relative affinity of the electron acceptors of the redox pairs isexpressed in terms of standard reduction potential, E0, (in volts) of theredox pair. That is, the potential of the reactions given in the reduction equationto occur. Let us learn about the standard reduction potential of the redox pairsand its significance.

7.5.2 Redox PotentialThe reactions in the redox couples given above are equilibrium reactions. Themagnitude of their equilibrium constants can provide information about theirrelative strength. However, handling electrons in such cases is an issue. Thesituation is similar to the one in which a metal is dipped into a solution of itsions. For example, if copper wire is dipped in a solution of copper ion (i.e.,copper electrode) the following equilibrium exists

…(7.53)

Similarly, for zinc metal dipped in a solution of zinc ion (i.e., zinc electrode) thefollowing equilibrium exists

…(7.54)

These two electrodes can be combined to form an electrochemical cell(Daniel cell). The relative strength of the electron acceptors (Cu2+ andZn2+ions) to accept the electrons would determine which of the two electronacceptors would accept the electrons and which of the two electron donors(Cu and Zn) would donate the electrons in the electrochemical cell. The


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potential difference between the two electrodes would depend on theirstandard electrode potentials. How do we know the standard electrodepotentials?

The standard electrode potential of these electrodes can be obtained bysetting up cells of these two electrodes individually with reference hydrogenelectrode, which has been assigned a potential of 0.00 V at all temperatures.The measured potential of the cell between a redox couple (under standardconditions; 25°C, solutes at 1 M concentration, gases at 101.3 kPa) andhydrogen electrode is called the standard reduction potential or standardredox potential. For zinc electrode,

…(7.55)

…(7.56)

You would recall from your earlier studies that the combination of a givenelectrode and hydrogen electrode is represented as following cell diagram.

…(7.57)

As a matter of convention, the standard hydrogen electrode is always taken asthe electrode on the left of the cell diagram and the electrode underconsideration on the right. The standard cell potential is always taken as

…(7.58)

Subtracting Eq. (7.56) from Eq. (7.55) we get the reaction as

…(7.59)

The measured value of the cell potential for cell reaction given in Eq. (7.59) isfound to be -0.77V. A negative value of Eo means that in this cell, zinc metalwould get oxidised to Zn2+ ions (i.e., zinc electrode would act as anode)whereas hydrogen ions would get reduced to hydrogen gas i.e., the following(and not the one given in Eq. (7.59)) reaction would occur spontaneously.

…(7.60)

Now if we replace the zinc electrode with copper electrode (under standardconditions) and measure the electrode potential we get a value of 0.34 V. Thisindicates that in this cell Cu2+ ions would get reduced to copper metal (i.e.,copper electrode would act as cathode) whereas hydrogen gas would getoxidised to hydrogen ions (i.e., hydrogen electrode would act as anode) i.e.,the following reaction would occur.

…(7.61)

Different redox couples are arranged in terms of decreasing value of theirstandard reduction potentials as given in Table 7.1.

A half-cell that takeselectrons from thestandard hydrogen cellis assigned a positivevalue of E0, and onethat donates electronsto the hydrogen cell, anegative value. ⇌⇌


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⇌

Zn(s)

Reduction half-reactions Eo, Volts

Oxidising agent Reducing agent

+2.65

+2.01

+1.23

+1.07

+0.77

+0.34

0.00

-0.25

-0.77

-1.66

-2.38

-3.04

In the event of an electrochemical cell constructed by combining anytwo electrodes; the electrode with a higher value of Eo would act ascathode and the one with a lower value would act as anode.

Going further, if we make a cell by combining zinc electrode and copperelectrode (i.e., make Daniel cell); there would be oxidation at the zincelectrode (anode, lower Eo) and reduction at the copper electrode (cathode,higher E0). The cell would be represented as

…(7.62)

The standard cell potential for the cell can be obtained by using Eq. (7.58).

…(7.63)

Having learnt about redox reactions and redox potential why don’t you assessyour understanding by solving the following simple question?

NADH can reduce pyruvate to lactate by the following reaction catalysed bylactate dehydrogenase.

Write this redox reaction in terms of two half-cells.

You may note here thatthe standard redoxpotentials do not giveany information aboutthe speed of thereaction.


Table 7.1:Standard reduction potentials (Eo) for some redox half-reactions at 298 K

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7.2.1 Nernst EquationThe discussion above has been on the standard reduction potential i.e., thereduction potential of a redox couple when all the species are at standardconditions (1 M concentrations). However, in real systems, the actual potentialof the redox couple would depend on the standard reduction potential and theactual concentrations of the species involved. The relationship between theactual reduction potential and standard reduction potential for a given redoxcouple was formulated by Walther Nernst, and is expressed in terms of anequation called Nernst equation according to which, for the electrodereaction:

…(7.64)

The reduction potential of the electrode at any concentration measured withrespect to standard hydrogen electrode is given by the following equation

…(7.65)

Where E is the standard electrode potential, R is the gas constant, T is

the absolute temperature, n is the number of electrons participating in thereaction, and F is the Faraday’s constant. The logarithm term is the ratio of theconcentrations of the reduced and oxidised forms of the species. As theconcentration (rather activities) for pure solids and liquids are taken as 1, theequation becomes,

…(7.66)

After converting natural logarithms to logarithm to the base 10, substituting thenumerical values for the constants, and assuming the temperature as 298K,we can write the equation as given below.

…(7.67)

For copper electrode, the Nernst equation at 298 K would be

…(7.68)

This is about one electrode or half-cell. In order to write the Nernst equation forthe cell, say Daniel cell, we would proceed as follows.

Anode: …(7.69)

Cathode: …(7.68)

The cell potential,

…(7.69)

…(7.70)

Mn+ /Mo


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…(7.71)

…(7.72)

Having learnt about the basic aspects of redox reactions in general, let us nowtake these up in the context of biochemical systems.

7.2.2 Standard Transformed Reduction Potential, E1°You have learnt above that the standard reduction potential refers to thereduction potential of a redox couple under standard conditions. Here,standard conditions refer to the physical chemistry standard state(concentration/activities=1, p=1 bar, T=298 K and so on). However, asdiscussed in the context of Gibbs energy for biochemical systems, we need toconsider biochemical standard state. Accordingly, the standard reductionpotential of a redox couple gets modified, and is called standard transformedreduction potential. Let’s take an example of a biochemical redox couple(fumaric acid/succinic acid) involving hydrogen ions to understand it

…(7.73)

The Nernst equation for the couple for certain concentrations of fumaric acidand succinic acid can be written as

…(7.74)

Under conditions of physical standard state the concentration (or activity) ofhydrogen ions would be 1. Substituting we get,

…(7.75)

However, under biochemical conditions the concentration of hydrogen ionswould be different from 1, and the potential would also be different. We canwrite,

…(7.76)

…(7.77)

…(7.78)

The first and the second terms on the right hand side equals .Substituting, we get

…(7.79)


E Fum/Succ E´fum/succ 0.059 pH

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Thus, for fumaric acid/succinic acid redox couple, the potential depends on thepH of the medium. However, if the succinic acid and fumaric acids are at unitconcentrations, and pH =7.0 (under biochemical standard state), then the Eq.(7.79) becomes

…(7.80)That is, the transformed standard reduction potential for the fumaric acid/succinic acid redox couple would be lesser suggesting lower tendency forfumaric acid to get reduced.

…(7.81)

This fact can also be rationalised as the effect of decreasing the concentrationof hydrogen ions (increasing the pH) on the equilibrium given in Eq. (7.73). Theequilibrium would shift to the left i.e., the tendency of fumaric acid to getreduced decreases. The standard transformed reduction potentials of somebiochemically important redox couples are given in Table 7.2.

Table 7.2: Standard transformed reduction potentials of somebiochemically important redox couples

Reduction half-reaction E’o/ V

Oxidizing agent Reducing agent

O2 + 4H+ + 4e–

Fe3 + (Cyt f) + e–

O2 + 2H2O + 4e–

Fe3 + (Cyt c) + e–

Fe3 + (Cyt b) + e–

Dehydroascarbic acid + 2 H+ + 2e–

Coenzyme Q + 2 H+ + 2e–

Oxaloacetate2– + 2 H+ + 2e–

Pyruvate– + 2H+ + 2e–

FAD + 2 H+ + 2e–

Glutathione (ox) + 2 H+ + 2e–

Lipoic acid (ox) + 2 H+ + 2e–

NAD+ + H+ + 2e–

2H2O + 2e–

Ferredoxin (ox) + e–

2H2O + 0.81

Fe2 + (Cyt f) +0.36

2H2O2 + 0.30

Fe2 + (Cyt c) + 0.25

Fe2 + (Cyt b) + 0.08

Ascorbic acid + 0.08

Coenzyme QH2 + 0.04

Malate2– - 0.17

Lactate– - 0.18

FADH2 - 0.22

Glutathione (red) - 0.23

Lipoic acid (red) - 0.29

NADH - 0.32

H2 + 2 OH– - 0.42

Ferredoxin (red) - 0.43

O2 - 0.45

Strongly reducing

E´ o Eo 0.059 7Fum/Succ fum/succ

E´ o Eo 0.413 VFum/Succ fum/succ


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7.5.5 Gibbs Energy Change and Reduction PotentialWe have seen above that the magnitude and the sign of the reduction poten-tials indicate the direction in which a redox reaction would proceed. Also, weare aware that the Gibbs energy change for a reaction also provides similarinformation. The question arises, "Are these two related?" The answer is 'yes',and in fact, it is a very significant relationship. The Gibbs energy change andthe standard reduction potential change for a redox reaction are related asfollows.

G nF E …(7.82)

G´O nF E´O …(7.83)

Where n is the number of electrons transferred in the reaction, and is thedifference in reduction potentials of the two half-cells as

…(7.84)

That is, we subtract the standard reduction potential of the half-cell undergoingoxidation from that of the half-cell undergoing reduction. In case of biochemicalsystems, we use the corresponding transformed reduction potentials.

Let us recall the example of fumaric acid/ succinic acid redox couple andrewrite the Eq. (7.81)

…(7.85)

Multiplying throughout by -2F, we get

…(7.86)

This gives

…(7.87)

Thus, the Gibbs energy change for the reaction under biochemical conditions(pH=7.0) is lesser negative, which means the reaction is more spontaneous atpH 0 than at pH 7.0.The relationships given in Eq. (7.82) and (7.83) can beconveniently used to determine the Gibbs energy change for a redox reaction,and hence calculate the corresponding equilibrium constant. Let us take anexample.

Example 3: In the terminal respiratory chain, the conversion of NADH to NAD+

generates two electrons, which are used to reduce molecular oxygen to water.The corresponding half-cell reactions are

The overall reaction is

The value of E’O can be calculated as

⇌


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From Eq. (7.83) we know that,

Substituting the values, we get

A part of this large amount of Gibbs energy is used for the synthesis of ATPmolecules.

Having learnt about redox reactions, redox couples and the related conceptswe are equipped to take up the universal electron carriers. However, beforemoving ahead, answer the following simple questions.

The half-reaction of the NAD+/NADH redox couple is given as under

Write the Nernst equation for the same.

7.6 UNIVERSAL ELECTRON CARRIERSAs discussed above, the redox reactions are accompanied by a change inGibbs energy. Further, the magnitude and sign of the standard reduction poten-tial (or transformed standard reduction potential) determine the magnitude andsign of the Gibbs energy change, and this in turn dictates the spontaneity of thereaction and the equilibrium constant. A great deal of energy transfer in biologi-cal systems happens through redox reactions, and is facilitated by electroncarriers. It is, therefore, pertinent to learn about the electron carriers and theirrole in biological systems. The electron carriers like, NAD+, NADP, FMN, andFAD undergo reversible oxidation and reduction in a number of the electron-transfer reactions in metabolism.

In the catabolic pathways, the energy released during the oxidation of nutrientsis picked up by the oxidised electron carriers, which in turn get reduced. Theenergy is captured in terms of the electrons in the reduced form of the electroncarrier. This energy becomes available when these electrons are eventuallytransferred to oxygen in the electron transport chain. Let us take the example ofoxidation of glucose to understand the role of electron carriers.

You know that the oxidation of glucose is the source of energy in many organ-isms. The aerobic oxidation of glucose to carbon dioxide and water can berepresented as

…(7.88)

A large amount of Gibbs energy is released in this process; the value of trans-formed Gibbs energy change G´O being 2840 kJ/mol. The overall reactioninvolves the oxidation of glucose and the reduction of oxygen. The correspond-ing half-reactions can be given as

…(7.89)

…(7.90)

⇌


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This means that a total of 24 electrons are transferred to 6 oxygen molecules.This transfer of electrons does not happen directly to oxygen in one step;instead these are transferred to the coenzymes NAD+ and FAD aided by anumber of enzymes of glycolytic pathway and Kreb's cycle to form 10 NADHand FADH2 molecules. These two reduced coenzymes viz., NADH and FADH2

carry these electrons and transfer them to oxygen in a meticulously organizedset of reactions in the electron transport chain. The transferred electronsparticipate in a series of redox reactions in four enzyme complexes beforereducing O2 to H2O. In the process of transferring the electrons to other spe-cies, the reduced coenzymes (NADH and FADH2) are reoxidised to NAD+ andFAD and become available for other reactions of substrate oxidation. Youwould learn in details about the electron transport in Unit 9 and 11. Let us beginwith NADH. You may recall from example 3 that NAD+ (the oxidised form ofNADH) gets reduced by taking up a proton and two electrons.


These electrons are eventually transferred to oxygen and the Gibbs energychange for the overall process is. This Gibbs energy is harnessed in astepwise manner as given in figure in the margin. Each of these three stepscontributes to the synthesis of ATP by oxidative phosphorylation. Overalloxidation of one molecule of NADH results in the synthesis of approximately2.5 molecules of ATP. NADH plays the role of electron carrier in a number ofreactions. In general, the reaction of NAD+ with the substrate (AH2) may begiven as:

…(7.91)

For example, the conversion of alcohol to aldehyde by alcohol dehydrogenasecan be given as

…(7.92)

On the other hand, the reduced coenzyme, FADH2 gets oxidised to FAD byCoenzyme Q (CoQ).

…(7.93)

The transformed standard Gibbs energy change for the process is -16.5 kJmol-1. This is not sufficient to synthesise ATP as it requires much more Gibbsenergy. The function of this reaction is just to inject the electrons from FADH2

into the electron transport chain. The details of the processing of electrons inthe electron transport chain and their eventual transfer to oxygen would betaken up in Unit 9. For now let us sum up what we have discussed in this unit.

⇌

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7.7 SUMMARYLet us recapitulate what we have learnt so far:

The study of the flow and transformation of energy in biological systems iscalled bioenergetics and in fact is a part of a bigger domain of study called'energetics' which refers to the study of energy in terms of its flow andtransformation within a system or between a system and its surroundings.

A 'system' in thermodynamics is defined as that part of the universe, whichis under consideration for the study, while the 'surrounding' in turn, is theremaining (other than the system) part of the universe. The system and itssurroundings put together constitute the universe.

Open system is such that it allows the transfer of matter and energybetween the system and its surroundings through it, while closed systemdoes not permit the transfer of matter but allows transfer of energy.

The first law of thermodynamics concerns conservation of energy i.e., inany physical or chemical process the total energy of the universe (system +surroundings) remains constant.

The Second Law of thermodynamics concerns the spontaneity or thedirectionality of a process. A spontaneous process is the one that occurs onits own under a given set of conditions without the aid of any externalagency.

Gibbs energy (G), named after American scientist Josiah Willard Gibbs, is ameasure of the net driving force of a process under the conditions ofconstant temperature and pressure.

Coupling reactions means reactions which are thermodynamicallyunfavorable (i.e., endergonic reactions) are combined or coupled with energyliberating (i.e., exergonic reactions), such that the Gibbs energy change foroverall reaction is negative and the process is exergonic (spontaneous).

Work done in living systems depends on flow of electrons in oxidation-reduction reactions occurring in them.

Standard reduction potential or standard redox potential is the potential ofthe cell measured between a redox couple (under standard conditions;25°C, solutes at 1 M concentration, gases at 101.3 kPa) and hydrogenelectrode.

The electron carriers like, NAD+, NADP, FMN, and FAD undergo reversibleoxidation and reduction in a number of the electron-transfer reactions inmetabolism. Two reduced coenzymes viz., NADH and FADH2 carry theseelectrons and transfer them to oxygen in a meticulously organized set ofreactions in the electron transport chain. This plays a significance role inharnessing and utilization of energy by biological systems.

7.8 TERMINAL QUESTIONS1) What are endergonic and exergonic reactions?

2) Why do we need to define a separate standard state for biochemicalreactions? What is biochemical standard state?


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3) Outline the need for non-equilibrium thermodynamics.

4) The biochemical reaction for hydrolysis of ATP is given below

Calculate the Gibbs energy change for hydrolysis of ATP in a celI having theconcentrations of ATP, ADP, and Pi as 3.4 mM, 1.3 mM, and 4.8 mM,respectively.

5) Standard Transformed Gibbs Energies of Hydrolysis of Creatine phosphateand ATP are given as under.

Calculate the transformed equilibrium constant for the reaction involvingtransfer of phosphate from Creatine phosphate to ADP.

6) Universal electron carrier, NADH is oxidised by oxygen as per the followingequation.

Write this redox reaction in terms of two half-cells.

7) Calculate the transformed standard Gibbs energy change, G´°, for thefollowing reaction involving reduction of acetaldehyde to ethanol by NADH

The E’ ° values for the NAD+/NADH and CH3CHO /CH3CH2OH redoxcouples are given as -0.320 V and -0.197 V respectively.

7.9 ANSWERSSelf-Assessment Questions

1) A thermodynamic system that allows the exchange of energy as well asmatter with the surroundings is called an open system, for example, foodbeing cooked in an open vessel. On the other hand, if the system permitsthe exchange of energy but not the matter it is termed as closed system,e.g., cooked food kept in a closed metallic container. The isolated systemdoes not permit the exchange of either the energy or the matter. The food inan insulated closed container is an example of an isolated system.

2) A spontaneous process is the one that occurs on its own under a given setof conditions without the aid of any external agency. According to the Sec-ond Law of thermodynamics for a process to be spontaneous, the totalentropy change for the system and surroundings ( Ssys + Ssurr) or uni-verse ( Stotal) must be positive i.e., Stotal = Ssys + Ssurr >0

3) Gibbs energy (G), is defined as G = H -TS. It is a measure of the net drivingforce of a process under the conditions of constant temperature andpressure and provides a criterion for spontaneity of a reaction.


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4) The Gibbs energy change for reaction

Can be given as

Under conditions of physical standard state (all species at 1Mconcentration)

rG = rGo …(A)

Now, under the conditions of biochemical standard state the expression forthe Gibbs energy change becomes

…(B)

Equating, Eq. (A) and Eq. (B), we get

Substituting the values of R and T, and converting natural log to the log tothe base 10, we get

Solving, we get

5) The coupled reactions refer to the coupling / combination of an endergonicreaction with an exergonic reaction such that the overall reaction isexergonic i.e., spontaneous. This is mediated through a commonintermediate between the two reactions, and is a consequence of the factthat the Gibbs energy is a state function. As an example, we can take theformation of ATP by oxidative phosphorylation from Creatine phosphate asper the coupling of following reactions

The overall reaction

is exergonic, and hence spontaneous.

6) To express a reaction as the difference of two reduction half-cells, first weidentify the species undergoing reduction and the product of reduction. Herewe see that pyruvate is getting reduced to lactate. The correspondingreduction half-reaction can be written (from Table 7.2) as

Pyruvate + 2H+ 2e– Lactate

In the next step we subtract this reaction from the overall reaction to get

NADH – H+ – 2e– NAD+


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This can be rearranged to give

This is the oxidation half-reaction, which can be reversed to give the corre-sponding reduction half-reaction

Thus the two half-reactions are as under

Pyruvate + 2H+ 2e– Lactate

7) The Nernst equation for the given half-reaction of the NAD+/NADH redoxcouple would be as follows

Terminal Questions1) Chemical (or biochemical) reactions accompanied by a decrease in Gibbs

energy ( G < 0) are spontaneous and are called exergonic, whereas theones associated with an increase in the Gibbs energy ( G > 0) are non-spontaneous and are called endergonic reactions.

2) The physical chemistry standard state for a reaction is defined as the onein which all the reactants and products are at unit activity (or 1M concentra-tion). For the reaction involving H+ ions, this corresponds to a pH=0. How-ever, as the biochemical reactions in the cell take place at a pH of about7.0, the physical chemistry standard state is not appropriate. We need toadopt a different standard state for biochemical reactions.

According to the biochemical standard state, the concentration of all thespecies are taken to be 1M as in physical chemistry standard state, but theconcentration of H+ ions is taken to be 1.0 x 10-7 M (i.e., pH = 7.0). Inaddition, the concentration of H2O is taken to be constant as 55.5 M and inthe reactions involving Mg2+ ions, their concentration is assumed to beconstant and taken as 1 mM = 1x10-3M, and the ionic strength, I=0.25.

3) A living cell is an open system having a continuous flux of energy andmatter into and out of it. At a given instance the cell is in a steady state i.e.,the concentrations of different constituents in it remain constant over aperiod of time. However, this continuous flow of matter across the cellularboundaries does not allow the establishment of a true equilibrium. The lawsof thermodynamics, which are for systems under equilibrium, are notcompletely applicable to the cell though they can provide certain details ofthe systems. Therefore, there is a need for non-equilibrium thermodynamictreatment for a better and detailed study/understanding of cellular reactions.

4) The Gibbs energy change for hydrolysis of ATP can be given as

It may be noted here that concentration of H2O does not appear in the


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expression as according to the biochemical standard state, it is constantand is included in the rG’ o term.

Now substituting the values of different terms, we get,

5) The reaction representing transfer of phosphate from Creatine phosphate toADP can be given as

This reaction can be obtained by adding reaction 1 and the reverse ofreaction 2 as follows

The corresponding Gibbs energy change would be

We know that: rG’ o = – RT In K’

Rearranging and simplifying:

Substituting the values we get, K’ = 4.63

know that the transformed Gibbs energy and reduction potentials arerelated as

G’ o = – nF E’o

substituting the values of n, F and E'o, we get

G’ o = –2(96500 J V–1mol–1) (0.123 V)

G’ o = –23.7 kJ mol–1

getting reduced to CH3CH2OH. The corresponding reduction reactions areas under

The E´ o value can be obtained by using Eq. 7.84 as

E´ o = E´ o (reduction) – E´ o (oxidation)

Substituting the given values of transformed standard reduction potentialswe get

E´ o = – 0.197 – (– 0.320) V = 0.123 V

rG 37700 Jmol 1 (8.314 JK 1mol 1)(298K)(2.303)log[1.3 10 3] [4.8 10 3]

[3.4 10 3]

⇌


6) In this reaction acetaldehyde takes two electrons from NADH, i.e., n=2. We

7) In this reaction NADH is getting oxidised to NAD+ whereas CH3CHO is

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We know that G´ o = –nF E´ o

Here, n=2; substituting the values, we get

G´O 2 (96500 Cmol 1)(0.123 V) 23.70 kJmol 1

7.10 SUGGESTED READINGSGarret, R.H., Grisham, C.M. (2016). Biochemistry (6th ed.). Boston,Cengage Learning. ISBN-10: 1133106293, ISBN-13: 978-1133106296

Berg, J.M., Tymoczko, J.L. and Stryer L., (2012) W.H. Biochemistry (7thed.), Freeman and Company (New York), ISBN:10: 1-4292-2936-5,ISBN:13:978-1-4292-2936-4.

Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7thed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611-6/ ISBN:10:1-6412611-9.

Lodish, H., Berk, A., Kaiser, C.A., Krieger, M., Bretscher, A., Ploegh, H.,Amon, A., Scott, M.P. (2016). Molecular Cell Biology (8th ed.). New York,WH: Freeman & Company. ISBN-13: 978-1-4641-0981-2.

Voet, D.J., Voet, J.G., Pratt, C.W. (2008). Principles of Biochemistry (3rded.). New York, John Wiley & Sons, Inc. ISBN:13: 978-0470-23396-2


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UNIT8

ATP AND HIGH ENERGYCOMPOUNDS

8.1 Introduction


8.2 ADP-ATP cycle

Hydrolysis of ATP

Synthesis of ATP

Processes that Produce ATP

8.3 Phosphorylation Potential

8.1 INTRODUCTIONIn the previous unit on Bioenergetics you learnt about the flow andtransformation of energy in biological systems, laws of thermodynamics, andGibbs energy changes. We also discussed about redox reactions as theseare one of the important means of energy transfer in living systems. A briefaccount of universal electron carriers and their roles and significance inharnessing and utilization of energy by biological systems was alsodiscussed.

All living organisms need energy for driving cellular processes. Cellularmetabolism constitutes of two kinds of biochemical reactions, catabolic andanabolic. Catabolic reactions liberate energy through reactions such asdegradation of carbohydrates. This energy eventually drives the energyconsuming anabolic processes, and also makes possible the other energy

8.4 Phosphoryl GroupTransfers

8.5 High Energy Molecules

8.6 Summary

8.7 Terminal Questions

8.8 Answers


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requiring process such as cell motility, muscle contraction, locomotion,transport of ions and substances against concentration gradients, conductionof nerve impulse etc. ATP has been recognized as the universal energycurrency of the cell because it is synthesized by using the energy liberatedfrom catabolic processes, and it is utilized to power the energy consumingprocesses which are essential for sustenance of life. In this unit we will learnabout the biochemically relevant high energy compounds such as PEP,creatine phosphate, ATP, ADP etc., and focus on the suitability of ATP as theuniversal energy currency in biochemical systems.


explain the ADP-ATP cycle;

describe the cellular processes that produce ATP and utilize it;

list high energy compounds;

explain the phosphorylation potential; and

describe the processes in the cell are driven by ATP hydrolysis and grouptransfer.

8.2 THE ADP-ATP CYCLEA small family of compounds universally mediates the flow of energy fromexergonic reactions to energy requiring processes in living systems. Thesehigh energy compounds are synthesized by utilizing energy released fromexergonic processes, and undergo hydrolysis (react with water) or oxidation(react with oxygen) making energy available to drive the energy requiringprocesses in living systems. NADH, FADH2, NADPH, ATP, ADP, SAM aresome of these high energy compounds. Reduced coenzymes, NADH, FADH2,NADPH, upon oxidation yield large amount of free energy (-150 to -220 kJ/mol). Adenosine triphosphate (ATP) is the most commonly utilized high energycompound which is actually a nucleotide comprising of D-ribose sugar,adenine base and three phosphate groups named as , and -phosphate(Fig. 8.1).

Fig. 8.1: The structure of ATP molecule.

Unit 8 ATP and High Energy Compounds

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There are several high energy phosphate compounds. ATP, ADP and AMP arephosphoric anhydrides. Phosphoenol phosphate (an enol phosphate) andacetyl phosphates (an acyl phosphate) are also categorized under high energyphosphate compounds. Creatine phosphate, a guanidino phosphate, andthioesters like acetyl CoA also yield large negative free energy upon hydrolysis.All these molecules transiently store chemical energy.

ATP hydrolysis yields -30.5kJ/mol energy (Fig. 8.2). ATP is the energy currencyof the cell. It is used for performing physical work such as muscle contractionand cell motility, chemical work such as biosynthesis of complexbiomolecules, and other works such as transporting ions and substancesacross membranes against concentration gradient.

Fig. 8.2: ATP hydrolysis liberates (–) 30.5 kJ/mol energy. Huge activation energyof 200-400 kJ/mol is needed for phosphoryl group transfer reaction. Suitableenzymes reduce this activation energy barrier (Garret and Grisham, 2017).

ATP is used as the primary energy currency in all living systems, frommicrobes to humans. The energy it carries is optimal for mediating mostbiochemical reactions. ATP is formed in phototrophic cells by photosynthesisand in heterotrophic cells by catabolism of other molecules. Hydrolysis of ATPto ADP and Pi (Fig. 8.3) provides energy for driving biosynthetic pathways andother work such as muscle contraction. The energy is stored in ATP moleculein the form of high energy phosphoric anhydride bonds.

On an energy scale, ATP is situated between extremely high energyphosphates and lower energy acceptor molecules. This unique position makesADP a good acceptor of energy and phosphates, while ATP can donate bothphosphates and energy to low energy acceptor molecules. Thus this ATP/ADPcouple is a good donor/acceptor system making it a versatile energy shuttleinteracting with various molecules giving or receiving energy.

Uninterrupted biosynthesis of ATP is critical for living cells, and any disruptionin this process is lethal to the cell. Poisons such as some cyanide compoundskill the cells by disrupting the process leading to ATP biosynthesis.


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8.2.1 Hydrolysis of ATP

Hydrolysis of ATP is a thermodynamically favorable reaction in the aqueousmilieu of living cells. ATP molecule contains two phosphoric acid anhydridelinkages (Fig. 8.4). Such linkages are seen in nucleoside diphosphates andtriphosphates, PPi etc. Hydrolysis reactions of these molecules are favorablereactions because of two reasons: i) the substrates (high energy molecules)have intramolecular repulsion because of multiple negative charges, and ii)the products are resonance stabilized.


Fig. 8.3: ADP-ATP cycle.

ATP powers most of the energy-consuming activities of cells (Fig. 8.5), someof which are as follows:

Most anabolic reactions including the biosynthesis of proteins, RNA andDNA by polymerization reactions from amino acids, ribonucleotides,deoxyribonucleotides respectively. All these processes require energy.

ATP provides energy for active transport of ions or molecules against theirconcentration gradient. Enzymes such as Na+/K+ ATPase, which transportions, consume most of the energy in human brain and kidney.

ATP is needed for bioluminescence in fireflies, which convert chemical

Fig. 8.4: Structure of ATP showing two phosphoric anhydride linkages.

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energy of ATP into light energy. Light flash is by virtue of luciferin which isactivated by luciferase involving ATP hydrolysis.

ATP is needed for muscle contraction. Myosin binds tightly to ATP andhydrolyses it. This drives the cyclic changes in conformation of myosin.Such conformational changes in many such myosin molecules causessliding of myosin fibrils along actin filaments, henceforth, causing musclefiber contraction.

Conduction of nerve impulses.

Phosphorylation of many different proteins which are required underdifferent physiological conditions including signaling.

ATP is also responsible for maintaining the pool of NTPs and dNTPs withinthe cells by the following reaction:

ATP + NDP (or dNDP) ADP + NTP (or dNTP)

One of the products formed in the reaction is ADP, which is used toreplenish ATP by the following reaction:

2ADP ATP + AMP

Beating of cilia and flagella in microorganisms for motility.

Maintenance of cell volume by osmosis, etc.

8.2.2 Synthesis of ATPPhosphorylases catalyze synthesis of ATP by phosphorylation of ADP by thefollowing reaction:

ADP +Pi ATP + H2O

It requires 7.3 kcal/mol energy, and occurs in the cytosol by glycolysis. Cellularrespiration occurring in mitochondria also synthesizes ATP. In plants, ATP issynthesized by photosynthesis in chloroplasts. Cells use ADP as precursorand add a phosphate group to it. Mitochondrial electron transport generates aproton gradient across the inner mitochondrial membrane. This is dissipated

Fig. 8.5: Synthesis and uses of ATP.


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through the F0F1 ATPase complex, and the energy released is utilized tophosphorylate ADP to produce ATP. The linkage between generation of protongradient and its dissipation to drive ATP synthesis is described bychemiosmotic coupling. This is why the F0F1 ATPase complex of mitochondriais also known as the coupling factor.

8.2.3 Processes that produce ATPATP is produced by three processes, namely, oxidative phosphorylation,photophosphorylation, and substrate-level phosphorylation (Figure 8.5). Allliving organisms produce ATP. In prokaryotes ATP is produced by glycolysis inthe cell wall and in cytosol. They can use sunlight, organic compounds suchas carbohydrates and even inorganic compounds as energy sources tosynthesize ATP. In bacteria, the ATPase and the electron transport chain arelocated within the cytoplasmic membrane. Breakdown of sugar and othernutrient molecules lead to accumulation of high concentration of the positivelycharged protons outside of the membrane than inside. This creates an excesspositive charge on the outside of the membrane and a relatively negativecharge inside. The result of this charge difference is dissociation of H2Omolecules into H+ and OH– ions. The H+ ions, produced in this manner, arethen transported outside of the cell and the OH– ions remain on the inside.This results in a potential energy gradient similar to that produced by charginga flashlight battery. The force the potential energy gradient produces is calledthe proton motive force (PMF) which can accomplish a variety of tasksincluding phosphorylation of ADP to ATP.

In eukaryotes, most ATP is produced in chloroplasts or in mitochondria. Inplants, ATP is synthesized in chloroplasts as well as in mitochondria, while inanimal systems most of the ATP is synthesized in mitochondria. Viruses, onthe other hand, do not have the machinery to synthesize their own ATP andhence they are called obligate parasites because of their dependence on hostfor its ATP requirements too.

Answer the following:

a) How much energy is liberated upon ATP hydrolysis?

b) ATP is situated between extremely high energy phosphates and lowerenergy acceptor molecules. What is the physiological relevance of thisintermediate position of ATP?

c) Name any three high energy phosphate compounds.

d) How many phosphoric acid anhydride linkages are present in a single ATPmolecule?

e) Reduced coenzymes are high energy compounds. Justify.

f) Why are cyanide compounds poisonous?

g) Name some processes in the cell which require ATP?


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8.3 PHOSPHORYLATION POTENTIALATP is the energy currency of cell. ATP links the catabolic and anabolicprocesses. It is utilized to make endergonic reactions feasible in the cell. ATPis converted to ADP and inorganic phosphate (Pi) or AMP and pyrophosphate(PPi) which involves group transfer reactions and hydrolysis. The ATPmolecule has four negative charges. Hydrolytic cleavage of –phosphateseparates two of these negative charges relieving the molecule from someintramolecular electrostatic repulsion.

ATP can also be a phosphoryl group donor. Under intracellular conditions (non-standard conditions), actual free energy of hydrolysis of ATP is known asphosphorylation potential, Gp. Under standard conditions, free energy ofhydrolysis of ATP is -30.5 kJ/mol. But this considers 1.0 M concentration ofATP, ADP and Pi. While in non-standard/cellular conditions:

i) The concentrations of ATP, ADP and Pi are neither same nor equal to 1M.

ii) These concentrations also vary from one cell type to another, and even inthe same cell type at different times.

iii) Also, Mg2+ is always present in cell shielding the negative charges of ATPand ADP. Therefore, in reality ADP and ATP exist in cells as MgATP2- andMgADP- (Fig. 8.6). So the phosphorylation potential, in true sense, is forMgATP2- hydrolysis and not just ATP hydrolysis. Inside the cell, ATP, ADPand Pi may also be found in states bound to proteins.

Fig. 8.6: The structure of Mg-ATP2- and Mg-ADP- (Nelson and Cox, 2017).

8.4 PHOSPHORYL GROUP TRANSFERSEnergy that ATP provides may not only be by simple hydrolysis, but also bymany group transfer reactions.

Direct hydrolysis of ATP involves its non-covalent binding followed by itshydrolysis providing energy for processes such as muscle contraction,translocation of enzymes along DNA/RNA molecule etc. The energy providedby direct ATP hydrolysis is also used for driving conformational changes inproteins required for different purposes.


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Group transfers

Phosphoryl (Pi), pyrophosphoryl (PPi) groups and AMP (adenylyl) groups aretransferred to substrate or amino acid residue of the enzymes by covalentlylinking these groups to enzymes/substrates. And in the second step, AMP/PPi/Pi becomes the leaving group (Fig. 8.7). And when these moieties aredisplaced, they contribute to free energy.

Fig. 8.7: ATP hydrolysis during group transfer reactions.

Answer the following:

a) Define phosphorylation potential?

b) Name the group transfer reactions in which ATP participates?

c) ATP molecule has intramolecular repulsion. How does it overcome thiselectrostatic repulsion?

d) Which processes in cells are driven by direct hydrolysis of ATP?

8.5 HIGH ENERGY MOLECULESPhosphate compounds can be divided into two groups:

i) High energy compounds have a G’° of hydrolysis more negative than-25 kJ/mol. For example: ATP ( G’°= -30.5 kJ/mol).

ii) Low energy compounds have a G’° of hydrolysis less negative than-25 kJ/mol. For example: glucose-6-phosphate ( G’°= -13.8 kJ/mol)(Table 8.1).


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Thus phosphorylated compounds have high or low phosphoryl group transferpotential (Fig. 8.8).

Fig. 8.8: High energy and low energy phosphate compounds and their standardfree energies of hydrolysis. (Nelson and Cox, 2017).

Catabolic reactions in living systems produce a number of high energyphosphate compounds, some of which are as follows:

Phosphoenolpyruvate (PEP)

Phosphoenolpyruvate has a high-energy phosphate bond (-61.9 kJ/mol).Hydrolysis of PEP by pyruvate kinase into pyruvate can phosphorylate ADP toATP (Fig. 8.9). In this manner the energy of PEP now becomes resident on anATP molecule. This chemistry is favorable since pyruvate is more stable thanPEP. PEP has only enol form while pyruvate has two tautomeric forms. Also,Pi is resonance stabilized. In plants, it is also involved in synthesis of a varietyof aromatic compounds and in carbon fixation; in bacteria, it is also used assource of energy for the phosphotransferase system.

Fig. 8.9: Hydrolysis of PEP. (Nelson and Cox 2017).


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1,3 - bisphosphoglycerate

1,3-BPG has the ability to phosphorylate ADP to form ATP. Its hydrolysis isaccompanied by a large, negative, standard free-energy change ( G´O = -49.3kJ/mol). The hydrolysis reaction yields 3-phosphoglyceric acid, which is thenconverted to resonance stabilized 3-phosphoglycerate (Fig. 8.10) favoring theforward reaction.

Fig. 8.10: Hydrolysis of 1,3-bisphosphoglycerate.

Phosphocreatine

In this molecule, the P-N bond can be hydrolyzed to generate free creatine andinorganic phosphate. Forward reaction is favored by the release of Pi and theresonance stabilization of creatine (Fig. 8.11). The standard free-energychange of phosphocreatine hydrolysis is -43.0 kJ/mol. Thus it is a high energycompound.

Fig. 8.11: Hydrolysis of Phosphocreatine.


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Thioesters

Thioesters are the product of esterification between a carboxylic acid and athiol. Here -S replaces the –O in the ester bond. Thioesters also have highnegative standard free energies of hydrolysis. Acetyl-coenzyme A (acetyl-CoA)is a well known thioester. Hydrolysis of this thioester (acetyl CoA) generates acarboxylic acid (acetic acid) which can ionize to carboxylate form (acetate)which is resonance stabilized. Hydrolysis of acetyl-CoA has G´O= -31 kJ/mol(Fig. 8.12; Table 8.1).

Fig. 8.12: Hydrolysis of Acetyl CoA.

Table 8.1: Standard free energies of hydrolysis of phosphorylatedcompounds

PEP (Phosphoenol pyruvate) G’° = -61.9 kJ/mol

1,3-BPG (1,3-bisphosphoglycerate) G’° = -49.3 kJ/mol

Phosphocreatine G’° = -43.0 kJ/mol

ADP AMP + Pi G’° = -32.8 kJ/mol

ATP ADP + Pi G’° = -30.5 kJ/mol

ATP AMP + PPi G’° = -45.6 kJ/mol

ATP AMP + PPi G’° = -45.6 kJ/mol

AMP adenosine + Pi G’° = -14.2 kJ/mol

PPi 2Pi G’° = -19.2 kJ/mol

Acetyl CoA G’° = -31.4 kJ/mol

Glucosee-6-phosphate G’° = -13.8 kJ/mol


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8.6 SUMMARY

Let us recapitulate what we have learnt so far:

High energy compounds are those whose oxidation or hydrolysis liberatesenergy more negative than -25 kJ/mol.

High energy compounds include PEP, creatine phosphate, ATP, ADP,Acetyl CoA etc.

ATP links the catabolic and anabolic reactions in the cells.

ATP is the energy currency of the cell. It is one of the high energycompounds.

There are multiple processes that utilize ATP. For example: Musclecontraction, active transport of ions, most anabolic reactions etc.

Various processes in cell produce ATP, namely, cellular respiration,photophosphorylation and substrate level phosphorylation.

ATP participates in a number of group transfer and direct hydrolysisreactions.

Group transfer reactions involve the transfer of phosphoryl,pyrophosphoryl and adenylyl moieties from ATP to substrate or amino acidresidues of proteins.

8.7 TERMINAL QUESTIONS1. Elaborate the cellular processes that use and synthesize ATP.

2. “Energy that ATP provides is not only by its hydrolysis but also by grouptransfer reactions.” Justify the statement.

3. In what aspects, standard and intracellular conditions vary and does itaffect the free energy of hydrolysis of ATP?

8.8 ANSWERSSelf-Assessment Questions1. a) Under standard conditions, ATP hydrolysis gives -30.5 kJ/mol.

However, under physiological conditions, the energy liberated upon ATPhydrolysis may vary from cell to cell due to the presence of metal ionsand, difference in concentrations of ATP, ADP and H+ from those understandard conditions.

b) ATP is situated between extremely high energy phosphate compoundsand lower energy acceptor molecules. This unique position makes ADPa good acceptor of energy and phosphates while ATP can donate bothphosphates and energy to lower energy acceptor molecules. Thus thisATP/ADP is a good acceptor and donor system making ATP a versatileenergy shuttle interacting with various molecules liberating or receivingenergy.

c) ATP, ADP, AMP, PPi, PEP etc.


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d) The ATP molecule contains two phosphoric acid anhydride linkages(Refer to Figure 8.4).

e) Reduced coenzymes, NADH, FADH2, NADPH, upon oxidation yieldlarge amount of free energy (-150 to -220 kJ/mol). Hence they areconsidered high energy compounds.

f) Few cyanide compounds are poisonous because they bind to thecopper atom in cytochrome oxidase which is one of the enzymesinvolved in ATP biosynthesis. The production of ATP is so crucial for cellsurvival that a poison which attacks any of the proteins used in ATPproduction would inevitably kill the organism immediately.

g) Active transport of ions and molecules, most anabolic reactions,bioluminescence, muscle contraction, conduction of nerve impulses,phosphorylation of many different proteins, maintenance of pools ofNTPs and dNTPs in the cell, cell motility by beating of cilia and flagella,maintenance of cell volume by osmosis (Refer to Figure 8.5) and manymore.

2. a) Under physiological conditions (non-standard conditions), the actualfree energy of hydrolysis of ATP is called as phosphorylation potential.

b) ATP participates in the transfer of phosphoryl (Pi), pyrophosphoryl (PPi)groups and adenylyl groups.

c) Mg2+ ions are always present within cells which shield the negativecharges of ATP and ADP. So in fact, ATP and ADP exist in cell asMgATP2- and MgADP respectively. This minimizes intramolecularelectrostatic repulsion.

d) In the cell, direct hydrolysis of ATP is involved in following processes:

muscle contraction,

movement of enzymes along DNA/RNA molecule

cycling of proteins between two different conformations.

Terminal Questions

1. Cellular processes that require ATP are: Active transport of ions andmolecules, most anabolic reactions, bioluminescence, musclecontraction, conduction of nerve impulses, phosphorylation of a number ofproteins, maintaining the of pool of NTPs and dNTPs within cells, beatingof cilia and flagella, maintenance of cell volume by osmosis (Refer to Fig.8.5) and many more. Cellular processes that synthesize ATP are cellularrespiration, photophosphorylation and substrate level phosphorylation.

2. Direct hydrolysis of ATP is used in few cellular processes like musclecontraction, movement of enzymes along DNA/RNA molecule, cycling ofproteins between two different conformations etc. ATP participates in avariety of group transfer reactions. It transfers Pi, PPi and AMP moieties toenzymes/substrates as the first step. In second step, these groups leavecontributing to free energy.


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3. Under intracellular conditions (non-standard conditions), actual freeenergy of hydrolysis of ATP is known as phosphorylation potential, Gp.Under standard conditions, free energy of hydrolysis of ATP is -30.5 kJ/mol. But this considers 1.0 M concentration of ATP, ADP and Pi each.While in non standard/cellular conditions:

i) [ATP], [ADP] and [Pi] are not same, and not equal to 1 M.

ii) These concentrations also vary from one cell type to another andeven in the same cell type at different times.

iii) Also, Mg2+ ions are always present in cell which effectively shield andcounter the negative charges of ATP and ADP. Therefore, in effect,ATP and ADP exist in cell as MgATP2- and MgADP- respectively. Sothe phosphorylation potential is in fact for MgATP2- hydrolysis ratherthan hydrolysis of just ATP. In addition, ATP, ADP and Pi often bind toproteins which may have its own influence.

Hence all these conditions occurring under the physiological conditions alterthe free energy of hydrolysis of ATP.



Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7thed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611-6 / ISBN:10:1-6412611-9.




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UNIT9

OXIDATIVEPHOSPHORYLATION

9.1 Introduction


9.2 Mitochondrial Electron Transport

Structure and Function of Mitochondria

Electron Transport Chain: Organizationand Function

9.3 Fo F1-ATP Synthase Complex

9.4 Peter Mitchell’s ChemiosmoticHypothesis

9.5 Energetics of Electron Transport

9.6 Regulation of OxidativePhosphorylation

9.1 INTRODUCTIONIn the previous unit you learnt about energy rich molecules. In this unit you willlearn about how these energy rich molecules are synthesized. You know thatmitochondria are considered as the “powerhouse” of the cell, since it is withinthis organelle that most of the energy for cells is generated. Mitochondriapossesses a system that couples respiratory electron transport chain tooxidative phosphorylation. Oxidative phosphorylation is the process in whichATP is formed as a result of the transfer of electrons from NADH or FADH2 toO2 through a series of electron carriers. Oxidative phosphorylation is

9.7 Inhibitors of ETC andUncouplers

Inhibitors of ElectronTransport Chain

Uncouplers

9.8 Thermogenesis

9.9 Summary

9.10 Term End Questions

9.11 Answers


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fundamental to cellular life in aerobic organisms since it is the major reactionfor generation of biochemically utilizable energy. In this unit, you will learnabout the electron carriers involved in the mitochondrial electron transport,generation of proton motive force, and how all these together account forbiosynthesis of ATP. You will also learn about regulation of oxidativephosphorylation, and inhibitors and uncouplers of this process.


explain the structural organization of mitochondria;

describe the structure and function of various components ofmitochondrial respiratory (electron transport) chain;

understand the organization of components of respiratory chain (electrontransport) into complexes;

explain the mechanism of oxidative phosphorylation;

explain chemiosmotic coupling hypothesis;

describe the energetic of oxidative phosphorylation; and

explain the action of inhibitors on respiratory chain and oxidativephosphorylation.

9.2 MITOCHONDRIAL ELECTRON TRANSPORTSince mitochondria are the site of oxidative phosphorylation, a goodunderstanding of the structural organization of mitochondria is required forappreciation of oxidative phosphorylation. You have already been providedsome details of mitochondria while studying TCA cycle in Unit 3 of BBCCT-109. Mitochondria are present in all aerobic eukaryotic cells, and they are thesite of the tricarboxylic acid cycle reactions, electron transport and oxidativephosphorylation. They are often localized near structures that require ATP,which is the major energy curency in biological sysems. For example, in theflight muscles of some insects, mitochondria are regularly arranged along themyofibrils. The ATP molecules formed by these mitochondria thus need todiffuse only to a short distance to the ATP-requiring contractile elements.Mitochondria are also frequently located adjacent to cytoplasmic fat droplets,which serve as a fuel source for synthesis of biochemically relevant form ofenergy, ATP. Let us now discuss the structural organization of mitochondria.

9.2.1 Structure and Function of MitochondriaThe most intensively studied mitochondria are those present in the liver cells.Under electron microscope, they appear as structures which are about 2 mmin length and less than 1 mm in width. Their size remains about the same inbacterial cells as well.

Mitochondria are a double membrane structure. As shown in Fig. 9.1, the outermembrane is smooth, while the inner membrane is invaginated to form foldsreferred to as the cristae. This increases the surface area of the inner

Mitochondria make up arelatively large fraction ofthe total cytoplasmicvolume which may varyfrom 20 to 50 percent ina given cell type.

Unit 9 Oxidative Phosphorylation

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membrane without a proportionate increase in the overall size of mitochondria.The internal mitochondrial matrix is proteinaceous and rich in enzymes.

The inner membrane ofliver mitochondriacontains at least 60different biologicallyactive proteins,embedded in thephospholipid bilayersystem. They includethe electron-transferringenzymes and proteins,enzymes involved inATP synthesis, variousdehydrogenases, andthe protein componentsof transport systems forvarious metabolites.Most of these proteinsare difficult to isolatefrom the membranestructure in functionalforms.

The two membranes of mitochondria differ in composition and biochemicalfunction. The outer mitochondrial membrane is permeable to many solutes butthe inner mitochondrial membrane is not. The inner mitochondrial membranecontains components of the electron transport chain and oxidativephosphorylation. It also contains components of shuttle systems that cantransfer electrons from cytosolic NADH to the mitochondrial electron transportchain, or from mitochondrial NADH to the cytosol. Hence, the rate of glycolysisin the cytoplasm and respiration in the mitochondria are integrated by theconcentration of ATP, ADP and phosphate in the cytosol and the mitochondria.Enzymes of TCA cycle and fatty acid oxidation are present in the mitochondrialmatrix. Table 9.1 lists some of the important mitochondrial enzymes and theirlocations.

Table 9.1: Location of certain enzymes in mitochondria.

Outer membrane Inner membrane

Monoamine oxidase NADH dehydrogenase (antimycin sensitive)

Kynurenine 3-monooxygenase Iron sulfur proteins

NADH dehydrogenase (antimycin- Cytochromes b, c, c1 and aa3 insensitive)

Acetyl-CoA synthetase F1 ATPase

Phospholipase A2 Succinate dehydrogenase

Nucleoside diphosphate kinase Carnitine acyltransferase

Matrix

Citrate synthase

Isocitrate dehydrogenase

Malate dehydrogenase

Fumarase

Aspartate transaminase

Glutamate dehydrogenase

Fatty acyl-CoA oxidation enzymes

Intermembrane space: Adenylate kinase

The outer membrane ofliver mitochondriacontains nearly 50 %lipids, whereas theinner membrane isdistinctive in containingonly about 20 % lipidsand nearly 80 percentproteins. The innermembrane ischaracteristically richin cardiolipin, whichmakes up about 20percent of the innermembrane lipids.


Fig. 9.1: Schematic representation of the structure of mitochondria.

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9.2.2 Electron Transport Chain: Organization andFunction

Mitochondrial electron transport chain is also referred to as the respiratorychain. It consists of four complexes namely, NADH-coenzyme Q reductase(Complex I), Succinate-Coenzyme Q reductase (Complex II), CoenzymeQ-Cytochrome c reductase (Complex III), and Cytochrome c oxidase(Complex IV). The sequential organization of these complexes of respiratorychain is depicted in Fig. 9.2. Transfer of electrons from NADH or FADH2 to O2

by a series of electron carriers generates 26 of the 30 molecules of ATP thatare formed when a glucose molecule is completely oxidized to CO2 and H2O.In this section we will discuss the organization and function of mitochondrialelectron transport chain.

The outer membranecan be removedexperimentally and theremainingmitochondrialstructure, consisting ofthe intact innermembrane plus matrix,is called mitoplast.

Metabolite transportacross the innermembrane ofmitochondria iscatalyzed by a nuclear-coded superfamily ofsecondary transportproteins calledmitochondrialtransporters/carriers.For example ATP-ADPtranslocase is one ofthe many mitochondrialtransporters engaged intransport of ions andcharged metabolites.

Fig. 9.2: Electron transport complexes of the inner mitochondrial membrane.

The mitochondrial electron transport chain utilizes NADH and FADH2 whichare energy-rich molecules generated during metabolic reactions includingglycolysis, fatty acid oxidation, and citric acid cycle. These energy-richmolecules have electrons with high transfer potential which are carriedthrough the electron transport complexes to O2. This transfer of electrons isalso accompanied by pumping of protons from the mitochondrial matrix intothe intermembrane space. This proton gradient across the inner mitochondrialmembrane generates a proton motive force, which is utilized to drive theendergonic reaction of phosphorylation of ADP to ATP.

Isolated mitochondria have also been demonstrated to carry out electrontransport under in vitro conditions. The inner mitochondrial membrane can beresolved into major electron transport complexes. This process may beundertaken in laboratory, and it involves mechanical treatment such assonication, selective solubilization using biological detergent such as digitonin,and separation methods such as centrifugation and chromatography. Fourmajor electron transport complexes have been isolated from innermitochondrial membranes which are shown in Fig. 9.2. These complexes, in away, function as multienzyme systems and together transport electrons fromNADH to O2. Now let us study the function of each mitochondrial electrontransport complex in detail.


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Respiratory Complex I

It is the first complex of mitochondrial electron transport chain which is alsoknown as the NADH-coenzyme Q reductase complex. It catalyzes the firststep of electron transport from NADH to CoQ. Complex I is an integral part ofthe inner mitochondrial membrane. It contains a molecule of flavinmononucleotide (FMN) and several (six to seven) iron-sulfur clusters whichparticipate in the electron transfer. This complex is the largest of all themitochondrial electron transport complexes.

The electron transport reaction occurs in several steps with successiveoxidation and reduction reactions. It begins with the transfer of electrons fromNADH to flavoprotein. In the next step, the reduced flavoprotein transfers itselectron to reduce the iron-sulfur protein while it gets itself reoxidized. In thethird step, the reduced iron-sulfur protein then donates its electrons tocoenzyme Q, which in turn gets protonated to CoQH2. Coenzyme Q is alsocalled ubiquinone. Transfer of two electrons from NADH to CoQ results intransport of four protons from mitochondrial matrix to the intermembranespace (Fig. 9.3).

NADH + H+ + CoQ NAD+ + CoQH2

Thirteen of the ~90polypeptides required foroxidativephosphorylation areencoded by mtDNA andsynthesized inmitochondria, whereasthe remaining proteinsare encoded by nuclearDNA, synthesized in thecytosol, and thentransported intomitochondria.

Fig. 9.3: Transfer of electrons from NADH to CoQ.

It may be noted that NADH can only participate in two-electron transfer,while both FMN and CoQ can accept and donate either one or twoelectrons. On the other hand, the cytochromes present in the Complex III,which receive electrons from reduced CoQ, can only undergo one-electronreductions. Therefore, FMN and CoQ mediate electron transfer between thetwo-electron donor NADH and the one-electron acceptors, the cytochromes.CoQ is a mobile electron carrier as it has a hydrophobic tail which allows it tobe present and be mobile within the lipid bilayer of inner mitochondrialmembrane.

Respiratory Complex II

It is also known as Succinate-CoQ reductase. It is the second of fourmembrane-bound complexes. It contains the dimeric citric acid cycle enzymesuccinate dehydrogenase and three other small hydrophobic subunits. Thiscomplex transfers electrons from succinate to coenzyme Q. The electrontransfer in this complex is facilitated by a covalently bound FAD, three Fe-Sclusters and one cytochrome b560. The substrate succinate (from the citric acidcycle) is oxidized to fumarate by a flavin enzyme succinate dehydrogenase, andelectrons are transferred to Fe-S clusters, and then to CoQ. No protonpumping across membrane is associated with this complex ie no protonsare pumped during this reaction.

Coenzyme Q, theuniquely mobilecomponent ofrespiratory chain, issoluble in the lipidcomponent of themitochondrialmembrane.


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Succinate + CoQ Fumerate + CoQH2

Respiratory Complex III

This complex is also called the CoQ-Cytochrome c reductase complex. It isthe third complex which is an integral part of the inner mitochondrialmembrane. Here the electrons received from reduced coenzyme Q arepassed on to cytochrome c in a multistep process. This complex contains twob type cytochromes, one cytochrome c1, and one Fe-S cluster.

Cytochromes are redox active proteins found in almost all organisms. Theycontain heme groups that reversibly alternate between Fe (II) and Fe (III)oxidation states during electron transport. The reduced cytochrome, whichhas Fe (II) oxidation state, exhibits prominent absorption spectra having threedistinct peaks, , and (Soret bands). The wavelength of the -peak variesand it is used for differentiating cytochromes. The -peak is absent in oxidizedcytochromes which have Fe (III) oxidation state. Mitochondrial membranesare known to have three types of cytochromes, cytochromes a,cytochrome b and cytochrome c. Slight differences may exist around theheme groups within each type of cytochrome. Therefore, each type ofcytochrome may further be divided into several other subtypes.

The complex III is located asymmetrically within the inner mitochondrialmembrane. Both cytochrome c1 and the non-heme iron-sulfur (Riske) proteinare located towards the outer surface, whereas cytochrome b spans throughthe membrane.

Cytochrome c is a peripheral membrane protein which is loosely bound to theouter surface of the inner mitochondrial membrane. It functions to shuttleelectrons from cytochrome c1 of Complex III and cytochrome c oxidase(Complex IV) by alternately binding them.

The overall reaction that takes place within this complex is

CoQH2 + 2 Cyt c [Fe (III)] CoQ + 2Cyt c [Fe (II)] + 2H+

As we have seen, coenzyme Q carried two electrons, whereas the reductionof Fe (III) to Fe (II) requires only one electron. Consequently, two molecules ofcytochrome c are required for every molecule of coenzyme Q.

Respiratory Complex IV

This complex is also called the cytochrome c oxidase complex. It catalyzesthe one-electron oxidation of four consecutive reduced cytochrome cmolecules, and the concomitant four-electron reduction of one molecule of O2.This is the final steps of mitochondrial electron transport.


4Cyt c [Fe (II)] + 4H+ + O2 4Cyt c [Fe (III)] + 2H2O

Like the other respiratory complexes, cytochrome oxidase is also an integralpart of the inner mitochondrial membrane and contains cytochromes a and a3,as well as two Cu ions (CuA and CuB) that are involved in the electrontransport process. The reduction of O2 to 2 H2O by cytochrome c oxidase


Leakage of electronsfrom the ETC producesreactive oxidationspecies (ROS) such assuperoxide (O–.),hydrogen peroxide(H2O2) and hydroxylradicals (OH.).

Presence of ROSdamage DNA and causelipid peroxidation.

2

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takes place on the cytochrome a3-CuB binuclear complex which involves fourconsecutive one-electron transfers from CuA and cytochrome a sites. Protonpumping across the inner mitochondrial membrane also takes place as aresult of this reaction.

The overall flow of electrons through the four complexes of mitochondrialelectron transport chain is summarized in (Fig. 9.4).

Fig 9.4: Flow of reducing equivalents through respiratory complexes (electrontransport chain) of mitochondria.

Here you must take a note that the mitochondrial electron transport chain isassociated with vectorial proton pumping across the inner mitochondrialmembrane which generates a proton gradient. The energy stored in the formof proton gradient is dissipated in an orderly manner to drive thephosphorylation of ADP to produce ATP. This is known as oxidativephosphorylation. The two processes electron transport and oxidativephosphorylation are coupled together which will be discussed in the comingsections.

The details of mitochondrial F0F1- ATP synthase complex are discussed in thenext sections.

Fill in the blanks:

a) (Outer/inner) ……………………….. mitochondrial membrane isinvaginated to form folds referred to as the cristae.

b) (Outer/inner) ………………… membrane is permeable to many solutes.

c) Components of the electron transport chain and oxidative phosphorylationare present on (outer/inner) ……………………….. mitochondrialmembrane.

d) Mitochondrial matrix contains enzymes of ……………………….(glycolysis/tricarboxylic acid cycle) and fatty acid oxidation.

e) Succinate dehydrogenase is present in ……………….. (mitochondrialmatrix/ chloroplast stroma).

f) Transfer of electrons from NADH or FADH2 to O2 by a series of electroncarriers generates ……… (18/26) of the 30 molecules of ATP.


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g) (Digitonin/Rotenone) …………………………. is used to resolve majorelectron transport complexes of the inner mitochondrial membrane.

h) (FMN/FAD) ……..………..) is a component of succinate dehydrogenasein electron transport chain.

i) Electron transport from NADH to CoQ is catalysed by ……………………(NADH-coenzyme Q reductase complex/Coenzyme Q).

j) ATP synthesizing system is located on.............. (outer/ inner)mitochondrial membrane.

k) (O2 /FAD)………………..has the highest redox potential.

Match the following

S.No. Column I S.No. Column II

a) Respiratory Complex I i Succinate-Co Qoxidoreductase/succinatedehydrogenase

b) Respiratory Complex II ii Cytochrome c oxidase

c) Respiratory Complex III iii CoQH2 cytochrome coxidoreductase/cytochromes reductase

d) Respiratory Complex IV iv NADH-CoQ oxidoreductase/NADH dehydrogenase

9.3 F0F1–ATP SYNTHASE COMPLEXThe energy stored in the form of proton motive force is conserved by driving theendergonic reaction of ATP synthesis from ADP and Pi. Fig. 9.5 is a schematicdiagram to depict the relationship between generation of H+ gradient across theinner mitochondrial membrane, and its dissipation to drive phosphorylation ofADP to produce ATP. This complex is also called the coupling factor, since itcouples the proton motive force to the process of ATP synthesis.

Fig. 9.5: A Schematic diagram showing connection between mitochondrialelectron transport and phosphorylation of ADP to give ATP.


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The F0F1-ATP synthase complex is constituted by two structurally distinctunits, each of which has several subunits. The F0 unit is located within theinner mitochondrial membrane and forms a transmembrane channel whichallows movement of protons along the concentration gradient. The role of F0

unit is in dissipation of the energy stored in the form of H+ gradient across theinner mitochondrial membrane. This energy is utilized by the F1 unit for ATPsynthesis.

The F0 unit consists of three hydrophobic subunits named a, b and c. Thesubunit stoichiometry is represented by a1b2c10-15, which means a single F0

unit has 1 molecule of the a subunit, 2 molecules of the b subunits, and 10 to15 molecules of the c subunit. The F1 unit of the F0F1-ATP synthase complexis spherical in shape. It is linked to the F0 unit and protrudes out from the innermitochondrial membrane into the mitochondrial matrix. The F1 unit consists offive different protein subunits called , , , , and . The subunit stoichiometryis represented as 3 3 (Fig. 9.6).

In 2016 Nobel Prize inChemistry was awardedto Jean-PierreSauvage, J. FraserStoddart, and Ben L.Feringa “for the designand synthesis ofmolecular machines”.Molecular machines aresingle-molecules thatbehave much like themachines peopleencounter every day:They have controllablemovements and canperform a task with theinput of energy.

Fig. 9.6: Structure of the F0F1 ATP Synthase complex of inner mitochondrialmembrane.

The purified F1 particles have been demonstrated to catalyze in vitro hydrolysisof ATP, a reaction which is the reverse of ATP synthase. For this reason, theF0F1-ATP synthase is also called the mitochondrial ATPase complex.

Elucidation of the mechanism by which movement of protons through the F0F1-ATP synthase complex drives the synthesis of ATP has been possible due tothe independent research works of John Walker and Paul Boyer. As depicted inFig. 9.6, the spherical part of the F1 unit which protrudes into the mitochondrialmatrix consists of three αβ dimers [( )3] each of which has a nucleotidebinding site. Although the three αβ dimers are identical, but their structuralstates, with respect to the nucleotide binding site, were found to be differentfrom each other at any given instance. It was suggested that one dimercontained ADP, the other contained ATP (which was actually in a derivetizednon-hydrolyzable form for allowing laboratory investigation), and the third wasempty. This protruding spherical part of F1 unit is connected to the F0 unit


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through the γ subunit, and the whole complex functions as a rotating engine(Fig. 9.7). Rotation of the subunit causes sequential conformational changesin the dimmers such that each dimer exists in one particular structuralstate. The three conformations of nucleotide binding site of the dimerswere named Loose (L), Tight (T) and Open (O) states. It has beenpostulated that ATP is formed spontaneously by condensation of ADP and Pi

without any energy requirement. The energy is actually required in thenext step when ATP has to be released from the T-state. This release ofATP from T-state is brought about by energy-dependent rotation of the subunit which creates a conformational change in the dimers. The ATPrelease is simultaneous to the binding of ADP and Pi to another dimer. Sothe first step is the passage of H+ through the F0 unit that powers rotation ofthe subunit by 1200 which opens the T site resulting into release of ATP. Itmeans that the T state has been converted to the O state. Now ADP and Pi

bind to the nucleotide binding site of the dimer which is in the O state. Inthe second step, passage of H+ through the F0 unit causes an additional 1200

rotation of the subunit. This brings the O state, which had just bound ADPand Pi, into the T-state where ATP is formed by their spontaneouscondensation. At the same time the O state, which had released ATP, bindsanother ADP and Pi and the next cycle starts all over again. For every fourprotons transported into the matrix one ATP is synthesized.

Fig. 9.7: The F1 unit of F0F1-ATP synthase functions as a rotating molecularmotor. Movement of H+ through the F0 unit fuels rotation of the v subunit of F1.This drives conformational change in the αβ dimers leading to synthesis andrelease of ATP as the proton gradient across the inner mitochondrialmembrane is dissipated.

9.4 CHEMIOSMOTIC COUPLING HYPOTHESISEnergy released by the oxidation reactions in mitochondrial electron transportchain is used to drive the synthesis of ATP. The coupling between electrontransport and oxidative phosphorylation has been explained successfully bythe British scientist Peter Mitchell in 1961. Mitchell’s theory is also known asChemiosmotic Coupling Hypothesis. The salient features of Mitchell’shypothesis are as follows:

Mitochondrial electron transport leads to proton pumping frommitochondrial matrix into the intermembrane space, thereby generating anelectrochemical H+ gradient (also known as proton motive force) acrossthe inner mitochondrial membrane.

The pH in the intermembrane space becomes lower than that in the

John Walker and PaulBoyer provided thestructural verification forbinding changemechanism for ATPsynthesis and wereawarded Nobel Prize forChemistry in 1997.

Boyer proposed therotational catalysismodel for ATPsynthesis.


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matrix, and this difference in pH is called ΔpH. In addition to the pHgradient, membrane potential also contributes to the electrochemical H+

gradient as expressed in the equation below:

H = - 2.3RT pH/F

Electrochemical Membrane pH gradientH+ gradient potential

In other words, the free energy spent in H+ pumping is stored in the form ofproton motive force. As the H+ concentration in the intermembrane spaceis higher, the protons have a tendency to flow back into the matrix, but thisis not possible since the inner mitochondrial membrane is impermeable toH+. This is the reason that generation and maintenance of proton motiveforce requires the inner mitochondrial membrane to be intact (Fig. 9.8).

The F0F1 ATP synthase complex, which is located in the vicinity of theelectron transport complexes, provides an opportunity for the protons toflow back from the intermembrane space to the mitochondrial matrix. Asthe protons flow back to the matrix, the energy stored as the proton motiveforce is dissipated. A part of this energy so released is utilized forsynthesis of ATP from ADP and Pi. Fig. 9.8 shows the electron transportand generation of electrochemical gradient across mitochondrialmembrane.

In other words the H+ pumping, that generates proton motive force, iscoupled to ATP synthesis. This is known as Chemiosmotic coupling asproposed by Peter Mitchell.

Fig. 9.8: Proton and electrochemical gradient across mitochondrial membrane.(Adapted from Garret and Grisham).


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Fill in the blanks:

a) The coupling between electron transport and oxidative phosphorylationwas given by ………………..

b) Mitchell’s theory is also known as ……………………………..

c) Electrochemical H+ gradient is also known as ………………………..

d) The F0F1 ATP synthase complex provides an opportunity for the protons toflow back from the intermembrane space to the …………………….

e) The …………….. unit of F0F1-ATP synthase complex forms atransmembrane channel which allows movement of protons along theconcentration gradient.

f) The ……………unit of the F0F1-ATP synthase complex is spherical inshape.

g) For every ……………… protons transported into the matrix one ATP issynthesized.

9.5 ENERGETICS OF OXIDATIVEPHOSPHORYLATION

The efficiency of oxidative phosphorylation can be measured if we are able tocalculate what percentage of total energy released by oxidation of a substratemolecule is conserved in the form of ATP. This efficiency is actually measuredas the P/O ratio which is defined as the number of molecules of ATPsynthesized per pair of electrons transferred through the mitochondrialelectron transport chain. In the laboratory, ATP synthesis is estimated byincorporation of phosphate into ATP, and number of electron pairs isquantitated by measuring O2 uptake which was reduced to H2O. Measurementof P/O ratios are subject to experimental errors which are difficult to avoid.The most widely accepted P/O values for NADH-linked and FADH2-linkedoxidations are 3 and 2 which may have errors. More recent measurementshave indicated two things, first that these values could be close to 2½ and 1½respectively, and second that the P/O values need not be integers. Thesenon-integer P/O values also indicate that oxidation and phosphorylation arenot directly coupled, rather there exists an indirect coupling between the twowhich does not require an integral stoichiometric relationship betweenreducing equivalents consumed and ATP synthesized. Now, if we considerthese values as correct, then the number of ATP synthesized per molecule ofglucose oxidized can be calculated as follows:

Glycolysis: Glucose + 2ADP + 2Pi + 2NAD+ 2 Pyruvate+ 2ATP + 2NADH + 2H2O + 4H+

Pyruvate 2 Pyruvate + 2NAD+ + 2CoA-SH 2 Acetyl-dehydrogenase complex: CoA + 2NADH + 2CO2


An integer is a wholenumber (not a fraction)that can be positive,negative, or zero forexample -1, 0, or +1.

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Citric acid cycle (including 2 Acetyl-CoA + 6H2O + 6NAD++ 2FAD +conversion of GTP to ATP): 2ADP + 2Pi 4CO2 + 6NADH + 2FADH2 +

2CoA-SH + 2ATP

Net: Glucose + 10NAD+ + 2FAD + 4H2O + 4ADP+ 4Pi 6CO2 + 10NADH +4H+ + 2FADH2 +4ATP

The total number of ATPs synthesized per molecule of glucose oxidized will be:

2½ ATP/NADH for 10 NADH/glucose +1½ ATP/FADH2 for 2 FADH2/glucose + 4ATP/glucose

i.e., 2½ X 10 + 1½ X 2 + 4 = 25+3+4=32

If the conventional values of P/O ratios of NADH as 3 and that of FADH2 as 2 areconsidered, then the total number of ATPs synthesized per molecule of glucoseoxidized will be 38. It is very important to note that there is a significantdisagreement over value of P/O ratios which are used in the above calculation.

It may also be noted that oxidation of NADH by O2 has a GO´ of -220 kJ/mol.

NADH + 4H+ + ½ O2 + 3 ADP +3 Pi NAD+ + 4 H2O + 3 ATP

Synthesis of ATP from ADP and Pi requires 31 kJ/mol energy under standardconditions. Therefore, the amount of energy conserved in the form of 3 ATPmolecules will be 3 x 31 kJ = 93 kJ. So as we see, out of a total of 220 kJenergy released, only 93 kJ (which is about 42% of the total energy) isconserved in the form of ATP under standard conditions.

9.6 REGULATION OF OXIDATIVEPHOSPHORYLATION

Oxidative phosphorylation is regulated by the availability of substrates. Themost important substrate is an oxidizable catabolite molecule (glucose) whichcould generate NADH or FADH2, or both. The availability of other substratesADP and Pi (for synthesis of ATP), and O2 as the terminal electron acceptor isequally important. Oxidative phosphorylation is absolutely dependent onmitochondrial electron transport since it has to generate proton motive forcewhich is essential for ATP synthesis. Continuation of electron transport wouldalso require an abundance of the terminal electron acceptor O2. In addition,ATP is synthesized only when it is required, and electron transport occurs onlywhen ATP is being synthesized simultaneously. This prevents any wastefuloxidation of glucose. This phenomenon is known as respiratory control inwhich utilization of glucose is controlled by ATP requirement.

Define the following terms:

a) P/O ratio

b) Proton gradient

c) Respiratory control

A reasonable mode ofaction for uncouplerscan be proposed in thelight of the existence ofa proton gradient.

Dinitrophenol is an acid;its conjugate base,dinitrophenolate anion,is the actual uncouplerbecause it can reactwith protons in theintermembrane space,causing a reduction indifference in protonconcentration betweenthe two sides of theinner mitochondrialmembrane.


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9.7 INHIBITORS OF ETC AND UNCOUPLERSOxidative phosphorylation is susceptible to inhibition at any point in theprocess. Several inhibitors have been identified which can block specificcarriers within the electron transport chain or in oxidative phosphorylation todisrupt the whole process of electron transport and ATP synthesis. Use ofthese inhibitors under laboratory conditions has yielded valuable informationon the sequence of organization of electron carriers in the respiratory chain,and the process of oxidative phosphorylation.

9.7.1 Inhibitors of Electron Transport ChainAlthough a number of inhibitors of electron transport chain are known, but thefollowing are worth mentioning:

i. Rotenone: It is a toxic plant product that was historically used by SouthAmerican Indians as a fish poison, and now commonly used as aninsecticide. It blocks the electron flow from NADH to CoQ.

ii. Amytal: It is a barbiturate drug, and acts at the same site as rotenone.

iii. Antimycin A: it is an antibiotic produced by Streptomyces griseus whichblocks electron transport from cytochrome b to cytochrome c1.

iv. Cyanide, azide, hydrogen sulfide and carbon monoxide: Theyfunction as cytochrome oxidase inhibitors. Cyanide and azide react withthe oxidized form whereas carbon monoxide reacts with the reducedform of the cytochrome target.

It is important to note that if an inhibitor of electron transport binds its targetelectron carrier, it disrupts the continuity of electron flow. As the electrontransport was continuing, all the electron carriers upstream of the target siteget into reduced state, whereas all the carriers which are situateddownstream of the target site remain in the oxidized state. In fact thisdifference in the redox state of carriers has allowed identification of the site ofaction of inhibitors.

9.7.2 UncouplersUncouplers are chemical compounds which inhibit phosphorylation withoutany disruption in mitochondrial electron transport chain. So the electrontransport continues right up to the terminal electron acceptor O2, but ATPsynthesis does not take place. The uncouplers function by dissipating the H+

gradient across the inner mitochondrial membrane which is generated due toelectron transport. In other words, they disrupt the coupling betweenelectron transport and oxidative phosphorylation. This loss of respiratorycontrol leads to increased oxygen consumption and oxidation of NADH. As aresult, metabolic fuel is continued to be consumed. Energy is not conserved inthe form of ATP, but it is released as heat. In fact, controlled uncoupling ofoxidative phosphorylation is the biological means of heat generation. Theprocess of uncoupling is reversible, and ATP synthesis resumes as theuncoupler is removed. 2, 4-dinitrophenol (DNP) is a common example ofuncouplers which functions by dissipation of the H+ gradient.

Proton-leak accounts fora significant part of theresting (basal) metabolicrate (BMR) and thus inmedical sciencesenhancement of thisprocess represents apotential target forobesity treatment.


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The antibiotic oligomycin is another phosphorylation inhibitor which binds to theF0 unit of the F0F1-ATP synthase complex and prevents the passage of H+ flowthrough it. This prevents the dissipation of proton gradient and, therefore, energyof H+ gradient could not be harnessed for ATP synthesis. The prevention of H+

gradient dissipation makes it increasingly more and more difficult for the electrontransport chain to pump protons into the intermembrane space. And eventuallyas a consequence, H+ pumping also ceases.

Give any two examples of:

a) Inhibitors of ETC………

b) Uncouplers of oxidative phosphorylation........

9.8 THERMOGENESISIn the above section you read about uncoupling of oxidative phosphorylationwhich leads to the generation of heat instead of ATP synthesis. This heat couldbe used to maintain body temperature. The coupling between mitochondrialelectron transport and oxidative phosphorylation is not perfect, and H+ leakageplays an important role in maintenance of basal metabolic rate (BMR) andbody temperature.

In a perfectly coupled system, protons enter the mitochondrial matrix onlythrough ATP synthase in the presence of ADP; and O2 is consumed only in thepresence of substrate (glucose) and ADP. However, F0 F1-ATPase-independent H+ leakage back into the matrix has been demonstrated inmammalian brown adipose tissue. This tissue is rich in mitochondria and lipiddroplets, and is a major source of non-shivering thermogenesis, used by mostmammals to resist low temperature. This function is mediated by a proteincalled UCP1 (uncoupling protein 1 or thermogenin). UCP1 is present in themitochondrial inner membrane and dissipates the H+ gradient by transportingprotons from intermembrane space into the matrix. This reduces the protonmotive force that drives ATP formation, and electron transport in the uncoupledmitochondria continues, releasing the energy only as heat. Another uncouplingprotein UCP3 is present in skeletal muscle and plays a similar role inthermogenesis in muscle tissue. This dissipative proton pathway is activatedby free fatty acids liberated from triacylglycerols in response to hormonessuch as thyroid hormone, epinephrine, and nor epinephrine.

9.9 SUMMARYLet us recapitulate what we have learnt so far:

Mitochondria are considered “powerhouse” of the cell. They possess asystem that couples respiratory electron transport chain to oxidativephosphorylation.

Oxidative phosphorylation is the process in which ATP is formed as aresult of the transfer of electrons from NADH or FADH2 to O2 through aseries of electron carriers.


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Mitochondria are a double membrane structure. The two membranes ofmitochondria differ in composition and biochemical function. The outermembrane is smooth, while the inner membrane is invaginated to formfolds referred to as the cristae which increases the surface area. Theinternal mitochondrial matrix is proteinaceous and rich in enzymes.

Mitochondrial electron transport chain also referred to as the respiratorychain consists of four complexes namely, NADH-coenzyme Q reductase(Complex I), Succinate-Coenzyme Q reductase (Complex II), CoenzymeQ-Cytochrome c reductase (Complex III), and Cytochrome c oxidase(Complex IV).

The mitochondrial electron transport chain utilizes NADH and FADH2 .Transfer of electrons is accompanied by pumping of protons from themitochondrial matrix into the intermembrane space which generates aproton motive force later utilized to drive the synthesis of ATP.

British scientist Peter Mitchell in 1961 successfully explained the couplingbetween electron transport and oxidative phosphorylation, also known asChemiosmotic Coupling Hypothesis.

The F0F1-ATP synthase complex couples the proton motive force to theprocess of ATP synthesis. For every four protons transported into thematrix one ATP is synthesized.

The efficiency of oxidative phosphorylation is measured in terms of P/Oratio which is defined as the number of molecules of ATP synthesized perpair of electrons transferred through the mitochondrial electron transportchain.

Rotenone, Amytal, Antimycin A, Cyanide, Azide, Hydrogen sulfide andcarbon monoxide, etc are some of the inhibitor of electron transport chainwhich can block specific carriers within the electron transport chain or inoxidative phosphorylation to disrupt the whole process of electrontransport.

9.10 TERM END QUESTIONS1. Describe the ultra structure of mitochondria.

2. What are the various components of mitochondrial electron transportchain?

3. Discuss the function of electron transport complex I.

4. Describe briefly the mechanism of oxidative phosphorylation.

5. Give a brief description of chemiosmotic mechanism involved in oxidativephosphorylation.

6. Explain the action of inhibitors on respiratory chain and oxidativephosphorylation.

7. What is thermogenesis? What is its significance?


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Block 1....................................................................................................................................................................................................................................

Biomembranes

9.11 ANSWERSSelf-Assessment Questions1. a) inner, b) outer, c) inner, d) tricarboxylic acid cycle, e) mitochondrial

matrix, f) 26, g) digitonin, h) FMN, i) NADH-coenzyme Q reductasecomplex, j) inner, k) O2

2. a) iv, b) i, c) iii, d) ii

3. a) Peter Mitchell in 1961, b) Chemiosmotic Coupling Hypothesis, c) protonmotive force, d) mitochondrial matrix, e) F., f) F., g) four

4. a) P/O ratio is the number of molecules of ATP synthesized per pair ofelectrons carried through the electron transport chain.

b) Proton gradient is the difference in hydrogen ion concentration acrossa membrane.

c) Respiratory control is the phenomenon which prevents any wastefuloxidation of glucose and is controlled by ATP requirement. ATP issynthesized only when it is required.

5. a) rotenone, amytal, hydrogen cyanide, etc

b) 2,4-dinitrophenol, oligomycin

Terminal Questions1. Refer section 9.2.1.

2. Refer section 9.2.2.


4. Refer section 9.3.






Nelson, D.L., Cox, M.M. (2017). Lehninger: Principles of Biochemistry (7thed.). New York, WH: Freeman and Company. ISBN: 13: 978-1-4641-2611-6 / ISBN:10:1-6412611-9.




BBCCT-111 MEMBRANE BIOLOGY AND BIOENERGETICS

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