Oxidative Phosphorylation

at the Inner Mitochondrial

Membrane (IMM):

Electron Transfer Pathway and

Thermodynamics of ET

Reactions (Reduction Potentials)

Dr. Ray

Chapter 18 - part 1 IN

OUT

http://higheredbcs.wiley.com/legacy/college/boyer/0471661791/animations/animations.htm

Oxidative Phosphorylation (Introduction)

2

Complete Glucose Catabolism Complete oxidation of glucose yields:

C6H12O6 + 6 O2 6 CO2 + 6 H2O DGo’ = - 2850 kJ/mol

The oxidation half of the overall reaction is:

C6H12O6 + 6 H2O 6 CO2 + 24 H+ + 24 e-

1. Electrons are EXTRACTED from glucose during which pathway(s)?

The reduction half of the overall reaction is:

6 O2 + 24 H+ + 24 e- 12 H2O

2. Electrons are delivered to oxygen during which pathway(s)?

• NAD+/NADH and FAD/FADH2 are cofactors that transfer electrons between glycolysis/citric acid cycle, and protein complexes at the IMM, yet they are present in SMALL amounts, so must be REGENERATED during ET.

• Average adult human uses 83 kg of ATP / day, but only has 250 g of ATP in body. Energy need is satisfied by recycling ADP back to ATP at rate of 300 x day, for each molecule of ATP.

3

Oxidative Phosphorylation: mitochondrial pathway that couples the

oxidation of NADH & FADH2 (produced by glycolysis of carbohydrates, b-oxidation of fatty acids, and the

citric acid cycle) to the phosphorylation of ADP to ATP, via a series of electron transfer reactions

in which O2 is the final electron acceptor

Extracting Energy via Electron Transfer Reactions

At the inner mitochondrial membrane NADH & FADH2 are REOXIDIZED to NAD+ & FAD:

NADH NAD+ + 2e- + H+

FADH2 FAD + 2e- + 2H+

1. Each reduced cofactor carries ____ e-

2. Where do all these electrons go?

3. How do cells harness the free energy of oxidation and use it to synthesize ATP?

Chemiosmotic Hypothesis:

Electron transfer is coupled to H+ flow, which is coupled to ATP synthesis

Electron Transport and Oxidative Phosphorylation

in Mitochondria

electrons

OUTSIDE

(cytosolic)

INSIDE (matrix)

H+cytosolic

H+matrix

1 NADHC yields ~

1 NADHM yields ~

1 FADH2 yields ~

IMM

Energy Conversion at the IMM 1. The overall process of electron transfer is____________, and the energy ________ during these ET reactions is utilized to _____ protons across the IMM. This is _________ H+ transport.

2. ATP is ___________ when H+’s flow back to the mitochondrial matrix through an enzyme complex called ATP synthase. This is ________ transport (since the protons are moving from high to low concentration). Energy released is ______________ to make ATP (an _____________ process).

3. How many complexes pump H+?

• ET complexes are active transporters, but energy for pumping comes from source other than ATP hydrolysis; it comes from spontaneous ET reactions, as carriers with LOW e- affinity release e- to those with HIGHER e- affinity.

• These are estimates and vary in different sources since ATP synthesis in the mitochondria is not stoichiometric, rather involves creation of a proton (H+) electrochemical gradient across the inner mitochondrial membrane (IMM).

• Oxidation of 1 glucose yields:

Oxidative Phosphorylation in Mitochondria

1 NADHC yields ~ 1.5 to 2 ATP

1 NADHM yields ~ 2.5 to 3 ATP

1 FADH2 yields ~ 1.5 to 2 ATP

• Of the 30 ATP formed when glucose is completely oxidized to CO2 and H2O, all but ___ come from oxidative phosphorylation.

10 NADH, 2 FADH2, 4 ATP = 30 to 36 ATP

We will use P:O ratio given in Stryer

in Table 18.4, (6th Ed. p. 531, and 7th Ed. p. 555),

yielding values below in blue (not those in gray)

P:O ratio = #ATP made per O atom reduced

electrons

- Number of ATP molecules produced by oxidative phosphorylation is variable, depending on how tightly coupled the inner mitochondrial membrane is (not leaky to H+).

7

(a) Glycolysis:

Net energy balance =

2 ATP + 2 NADH

(b) Pyruvate dehydrogenase

complex:

(c) Citric Acid Cycle:

2 ATP + 6 NADH

+ 2 FADH2

1 (mitochondrial) NADH yields ~ 2.5 ATP

1 FADH2 yields ~ 1.5 ATP

1. How much total energy is released by complete catabolism of ONE glucose?

2 NADH

TCA extracts:

PDHC extracts:

• Aerobic conditions: 1 glucose molecule can yield upto ~ 30 ATP molecules

3

1 (cytosolic) NADH yields ~ 1.5 ATP

5

15

3

Overview of Energy Captured from Oxidative Catabolism of One Molecule of Glucose

cytosol

mitochondria

2 turns of TCA

Aerobic

Glycolysis can yield

8

Sequence of Electron Transfer Reactions

• Energy for ATP synthesis comes from spontaneous ET reactions, as electrons are transferred from carriers with LOW electron affinity (high electron transfer potential) to those with HIGHER electron affinity (low ET potential).

Complex I

Complex III

Complex IV

Path of electrons delivered by reduced cofactors to Oxygen: What is the basis for this electron flow pathway? (Why do e- move in this order?)

Different molecules have different affinities for electrons. This is termed the REDUCTION POTENTIAL of the molecule

IN

OUT

4 H+ 4 H+

2 H+

http://www.wiley.com/college/fob/quiz/quiz17/17-8.html

• O2 has most positive Eo’ (highest affinity for electrons), so once electrons have reached O2, they are in their lowest energy (most stable) state.

• As electrons are transferred from NADH to O2, they are moving to lower energy states, so energy is RELEASED as electrons move through the electron-transport chain!

DEo’ is proportional

to DGo’

Energy Level Diagram:

DGo’ = - n F DEo’ -

+

Standard Midpoint Reduction Potentials

• Likewise, molecules that undergo redox reactions have a characteristic strength as an electron donor ! This is expressed as the midpoint potential (Eo') at pH 7, which is measured in the unit of volts.

• Standard midpoint reduction potential Eo’ is a measure of a substance’s affinity (or attraction) for electrons.

1. In which direction will electrons flow spontaneously if the species shown in the half reactions above are combined? WHY?

because O2 has a _____________ (attraction) for electrons (more positive Eo’)

• The strength of a weak acid is expressed by its Ka (or pKa) value, which is a measure of its strength as a proton donor.

• A negative reduction potential means that the reduced form of a substance has lower affinity for electrons than does H2. • A positive reduction potential means that the reduced form of a substance has higher affinity for electrons than does H2. These comparisons refer to standard conditions: 1 M oxidant, 1 M reductant, 1 M H+, and 1 atmosphere H2

• The standard midpoint potential (reduction potential) is the voltage at which the molecules are half oxidized and half reduced:

Cyt c (Fe3+) + e- Cyt c (Fe2+) Eo' = + 0.22 V

½ O2 + 2 e- + 2 H+ H2O Eo' = + 0.82 V

direction of e- flow (towards more positive redox potential)

corresponds to negative

DGo’ (-)

e- donor

e- acceptor

11

Standard Reduction Potentials

http://www.nature.com/nrm/journal/v3/n11/images/nrm954-i2.gif

During the ET chain, a strong reducing agent (such as NADH) with a negative reduction potential, is poised to donate electrons,

whereas a strong oxidizing agent (such as O2) with a positive reduction potential is ready to accept electrons.

1. REDUCTANT: The species with the most negative standard midpoint potential is the strongest electron donor (and weakest electron acceptor).

2. OXIDANT: The species with the most positive standard midpoint potential is the weakest electron donor (and strongest electron acceptor).

3. Electrons flow spontaneously from species with LOW e- affinity (most negative, or least positive reduction potential) to ones with HIGHER e- affinity (most positive reduction potential).

4. Use Eo’ to predict if a given species can reduce another.

12

Thermodynamics of Electron Transport Sequential electron transport through transmembrane complexes

I, III, IV involves the overall exergonic reaction:

NADH + ½ O2 + H+ NAD+ + H2O DGo’= -220 kJ/mol

Q: Calculate the change in reduction potential (DEo’) and the driving force (DGo’) of the reaction above using the information given below.

NAD+ + H+ + 2e- NADH DEo’ = - 0.32 V

½ O2 + 2 H+ + 2e- H2O DEo’ = + 0.82 V

DEo’ = Eo’electron acceptor - Eo’electron donor

Strategy: Determine which reactant is the e- donor and e- acceptor:

1. Is NADH oxidized or reduced in overall reaction?

• Since NADH is oxidized, it is loosing electrons, so it is the electron donor (it is the reducing agent or reductant, with least positive DEo’)

2. Is O2 oxidized or reduced in overall reaction?

• Since O2 is reduced, it is gaining electrons, so it is the electron acceptor (it is the oxidizing agent or oxidant, with most positive DEo’)

13

• n is the number of moles of electrons transferred in the reaction: (n = 2e- for NADH) • F is Faraday's constant: 23.06 kcal/mol.V = 96.5 kJ/mol.V

• The superscript o corresponds to values at standard conditions (T = 298 K, all gases are at 1 atm, concentrations of all relevant species = 1.0 M).

• Except [H+] = 1 x 10-7 (pH=7)

DGo’ = - n F DEo’

Thermodynamics of Electron Transport

-

+

This is related to the change in free energy by the Nerst Equation:

DEo’ = Eo’electron acceptor - Eo’electron donor

Calculate the standard change in reduction potential:

• When DEo’> 0 (positive), then DGo’ < 0, and the reaction is a spontaneous electrochemical reaction in the forward direction as written.

• When DEo’ < 0 (negative), then DGo’ > 0, and the forward reaction is NOT spontaneous, and the electrochemical reaction in the reverse direction will occur.

14

Thermodynamics of Electron Transport: NADH

• Overall ET through transmembrane complexes I, III, IV involves:

NADH + ½ O2 + H+ NAD+ + H2O oxidized reduced e- donor e- acceptor

DEo’ = Eo’electron acceptor

- Eo’electron donor

DEo’= __________

DGo’= __________ Calculate DEo’ and DGo’:

Use numbers as given in table, do NOT switch sign for e- donor in

calculation!

This potential difference between NADH and O2 drives ET.

1. Is electron transfer from NADH to O2 energetically favorable?

DGo’ = (- 2)(96.5kj/mol.V)(1.14V) = - 220 kJ/mol

= 0.82 – (-0.32 V) = 1.14V

DGo’ = - n F DEo’ -

+

15

1.

2.

3. Which is the strongest reducing agent?

4. Which is the strongest oxidizing agent?

16

Thermodynamics of Electron Transport: FADH2

FADH2 + ½ O2 + 2H+ FAD + H2O

DEo’ = Eo’electron acceptor - Eo’electron donor

DGo’ = - n F DEo’

DEo’= _________

DGo’= _________

1. Using information in Table 18.1 calculate DEo’ and DGo’ for FADH2 oxidation in reaction below:

Use numbers as given in

table, do NOT switch sign

for e- donor, since formula

already has a negative sign.

For FADH2, overall ET occurs ONLY thru transmembrane complexes III & IV

during rxn:

Find redox potentials in Table 18.1 for reactants in overall reaction: FAD + H+ + 2e- FADH2 DEo’ = - 0.22 V ½ O2 + 2 H+ + 2e- H2O DEo’ = + 0.82 V

reducing agent oxidizing agent

17

Reduction Potentials of Electron Transport Chain Components in Mitochondria

During ET at the IMM, electrons flow spontaneously and sequentially from species with lower affinity to species with higher affinity for electrons!

+ n e-

When FADH2 is oxidized, is more or less energy

released than oxidation of NADH? Why?

e- donor

e- acceptor

Information in Table is always written: oxidant + e- reductant

even though the reaction with the lowest DEo’ runs in reverse!

e- donor (most neg. Eo’)

= species oxidized (this rxn runs in reverse)

e- acceptor (most pos. Eo’)

= species reduced (this rxn runs forward)

Ubiquinol

18

Chemiosmotic Hypothesis

2. This flow of electrons leads to pumping of protons (H+) out of the mitochondrial matrix into the inter-membrane space.

3. As H+s flow back into the matrix, ATP is made by ATP synthase.

Q: Is there a high-energy intermediate that couples electron flow to ATP synthesis, like 1,3-BPG (which has phosphoryl group transfer potential) or succinyl CoA (which has acetyl group transfer potential) ?

1. Electron Transport Chain (ET) involves the flow of electrons from organic metabolites to O2 via the redox carriers of the respiratory chain, through four large integral membrane protein complexes I to IV that are embedded in the IMM (Inner Mitochondrial Membrane).

Several energy transformations

occur during

oxidative phosphorylation

Energy Conversion at the IMM

1. How many complexes are involved in oxidative phosphorylation?

2. Where is ATP synthesized?

3. How is ATP synthesized?

Oxidative Phosphorylation is: oxidation (of NADH and FADH2) coupled to phosphorylation (of ADP)

http://www.genome.ad.jp/kegg/pathway/map/map00190.html

IN

OUT

____________________________ drives a “primary transporter to work in reverse”

1o transporter = ATP hydrolysis drives H+ pumping ATP synthase = H+ flow drives ATP synthesis

Path of electrons delivered by NADH to Oxygen: NADH Complex I CoQ complex III cytochrome c complex IV

(Cytochrome c oxidase) O2

20

Electron Transport Sequence in Mitochondria 1. What is the path of electrons from oxidation of 1 glucose? Which

pathway/steps provide reduced cofactors, and how many electrons come from each pathway?

Both: CoQ complex III cytochrome c complex IV O2

NADH: Complex I CoQ Glycolysis (2), Pyruvate Dehydrogenase (2) Citric Acid Cycle (6)

FADH2: Complex II (succinate dehydrogenase) CoQ Citric Acid Cycle (2) Succinate Fumarate

2.5ATP 1 NADHM

1.5ATP 1 FADH2

[total: 10 NADH]

_____ H+ pumped per NADHM (electrons pass through Complexes I, III, IV)

_____ H+ pumped per FADH2 (e- only pass through III & IV - bypasses I)

2. How much energy is captured for 2e- delivered by each cofactor?

[total: 2 FADH2]

21

Concentration Gradients and Free Energy

• Electron transport of 1 molecule of NADH results in the pumping of 10 protons (H+) across the IMM, and in the proton-electrochemical gradient (involves concentration & electrical potential terms).

2. How many molecules of ATP can be synthesized as these protons flow back into the matrix?

Since _____ kJ/mol is required to synthesize ATP from ADP + Pi ,

_____________ ATP could be synthesized per NADH oxidized.

• Some of the energy is lost as heat, so 2.5ATP are actually made per NADH oxidized.

• Overall efficiency of oxidative phosphorylation is:

~ 42% in standard biochemical conditions (DGo’)

~ 70% in physiological concentrations, (DG’)

• 3 complexes pump protons

• 10 H+ pumped per NADH

1. How much free energy is stored in the H+ gradient across the IMM?

storage of ~ 220 kJ/mol of free energy

http://www.neuro.wustl.edu/neuromu

scular/pics/diagrams/elecflow2.gif

Electron Transport Sequence • Complexes named after species oxidized (electron donor) and species

reduced (electron acceptor)

1. If respiration is blocked at different points along the ET chain, will ATP synthesis continue? Why?

all ET stops so H+ pumping stops once H+ gradient is used up, no more forms ATP synthase has no energy source to make more ATP

You need to know these names & functions!

Oxidoreductases: Oxidize one cofactor and transfer these

electrons to another cofactor (reduce it)

(FAD)

(FADH2)

(a) I

(b) II

(c) III

ET can occur thru other path

23

Electron Transport (ET) Sequence for NADH

• Complexes I, III, IV are transmembrane proteins that utilize the energy released by the flow of electrons through their redox centers (- DG) to pump protons (H+) from the matrix (IN) to the cytosolic (OUT) side of the IMM.

• Complexes I, II, III, IV have organic or inorganic prosthetic groups (redox centers) that actually bind & transfer electrons through the protein:

• Mobile carriers shuttle electrons between integral membrane complexes:

5. Quinone (organic CoQ - lipophilic) 6. Cytochrome c (globular metalloprotein - hydrophilic exterior)

NADH Complex I CoQ complex III cytochrome c complex IV (Cytochrome c oxidase) O2

IN

OUT

Inner Mitochondrial Membrane (IMM)

1. flavins (FMN)

2. hemes (cytochromes, Fe3+/Fe2+)

3. iron-sulfur clusters (FeS)

4. copper ions (Cu2+/Cu+)

Each complex has multiple redox centers, so several ET reactions occur within each complex.

24

25

Sequence of Electron Transport Chain Components

1. During mitochondrial ET, how do we know the order of electron transfer reactions?

½ O2 + 2H+ + 2e- H2O

Eo’ = + 0.815V (highest e- affinity)

• Compare the standard reduction potentials Eo’ of the redox cofactors, since this indicates their affinity (attraction) for electrons.

So in ET at the IMM, electrons flow sequentially to species with higher

affinities for electrons!

Electrons flow sequentially from species with a lower affinity for electrons (usually at top of table, more negative Eo’) to species with higher affinity for electrons (usually lower in table, more positive Eo’).

http://higheredbcs.wiley.com/legacy/college/voet/0471214957/guided_ex/electron_transport/electron_transport.html INTRODUCTION

26

Thermodynamics of Electron Transport (ET)

Energy level diagram: shows flow of electrons among components of the ET chain: from species with lower e- affinity (at top of diagram) towards species higher e- affinity (at bottom)

Overall reaction is: NADH + ½ O2 + H+

NAD+ + H2O

1. Is there enough energy to synthesize ATP?

NADH oxidation provides

enough energy for the synthesis of several molecules of ATP:

DGo’= ____ kJ/mol

ADP + Pi ATP + H2O DGo’ = ______ kJ/mol

How many electrons are needed to fully reduce 1 molecule of O2? How many molecules of NADH are needed to deliver these e- ?

28

ET Problem Analysis of the electron transport pathway in a pathogenic gram-negative

bacterium reveals the presence of five electron transport molecules with the redox potentials:

A) Predict the sequence of the carriers in the electron transport chain.

B) How many molecules of ATP can be generated under standard condtions when a pair of electrons is transported along this pathway?

C) Why is it NOT likely that oxygen is the terminal electron acceptor in this bacterium? The redox potential for ferroprotein __________ is more positive than that of O2 ____________

DEo’ = _____________________

DGo’ = __________________________________________

Table 2: