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Short Review

Listening to PS II: Enthalpy, entropy, and volume changes

Harvey J.M. Hou a,⇑, David Mauzerall b,⇑a Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, 285 Old Westport Road, North Dartmouth, MA 02747, United Statesb The Rockefeller University, 1230 York Avenue, New York, NY 10065, United States

a r t i c l e i n f o

Article history:Available online 15 March 2011

Keywords:ThermodynamicsPS IIPhotoacousticsEnthalpyEntropyVolume change

a b s t r a c t

Photosystem II, located in the thylakoid membranes of green plants, algae, and cyanobacteria, uses sun-light to split water into protons, electrons, and a dioxygen molecule. The mechanism of its electron trans-fers and oxygen evolution including the structure of the protein and rates of the S-state cycle has beenextensively investigated. Substantial progress has been made; however, the thermodynamics of PS IIelectron transfer and of the oxygen cycle are poorly understood. Recent progress in thermodynamic mea-surements in photosynthesis provides novel insights on the enthalpic and entropic contribution to elec-tron transfer in proteins. In this review the thermodynamic parameters including quantum yield,enthalpy, entropy, and volume changes of PS II photochemistry determined by photoacoustics and otherlaser techniques are summarized and evaluated. Light-driven volume changes via electrostriction aredirectly related to the photoreaction in PS II and thus can be a useful measurement of PS II activityand function. The enthalpy changes of the reactions observed can be directly measured by photoacous-tics. The apparent reaction entropy can also be estimated when the free energy is known. Dissecting thefree energy of a photoreaction into enthalpic and entropic components provides critical informationabout mechanisms of PS II function. Potential limitations and future direction of the study of the thermo-dynamics of PS II electron transfer and oxygen evolution are presented.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3572. Overview of photoacoustic measurements in photosynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3593. Thermodynamics of charge separation, electron transfer, and S-state cycle in PS II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

3.1. Quantum yield. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3593.2. Molecular volume changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3603.3. Enthalpy changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3613.4. Entropy changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3623.5. Comparison of the thermodynamics of bacterial, PS I, and PS II centers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

4. Limitations and potential problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3635. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

1. Introduction

Oxygenic photosynthesis involves the cooperation of twophotosystems (PS), PS I and PS II in cyanobacteria, algae, and higher

plants [1,2]. PS II is an integral membrane protein complex locatedin the thylakoid membrane and utilizes the energy of sunlight tosplit water into protons, electrons, and molecular oxygen. In thepast ten years the three dimensional structure of PS II was deter-mined at 3.8 A from Synechococcus elongates [3] and 3.0–3.7 A fromThermosynechococcus elongates [4–6]. The crystallographic modelprovides complete arrangement of 70 cofactors and protein matrixof 20 protein subunits in the structure of PS II. As shown in Fig. 1,these cofactors include the primary electron donor (P680), primary

1011-1344/$ - see front matter Published by Elsevier B.V.doi:10.1016/j.jphotobiol.2011.03.007

⇑ Corresponding authors. Tel.: +1 508 999 8234; fax: +1 508 999 9167 (H.J.M.Hou), tel.: +1 212 327 8218 (D. Mauzerall).

E-mail addresses: hhou@umassd.edu (H.J.M. Hou), mauzera@rockefeller.edu(D. Mauzerall).

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electron acceptor pheophytin (Pheo), quinone secondary acceptors(QA and QB), and tyrosine electron donor (YZ). The oxygen evolutionMn4Ca center is composed of a Mn3CaO4 cuboid with the fourthmanganese ion attached to a corner of the cuboid. The so called‘‘dangler’’ Mn ion and Ca ion may be the active site for water oxi-dation [7].

The kinetics of the electron transfer reactions in PS II has beenthoroughly investigated over the complete timescale of femtosec-ond to seconds [8–10]. Fig. 2 shows the general pathway and elec-

tron transfer rates in PS II. The early processes occurring in PS IIrevealed by ultrafast spectroscopy can be divided into severalsteps: (a) absorption of light quanta by antenna to form excitedstates of pigments; (b) trapping of excitation energy by the pri-mary electron donor P680 in the reaction center on the picosecondtime scale; (c) primary charge separation from the singlet excitedstate of P680 to the primary acceptor Pheo in about 3–20 ps; (d) sta-bilization of the separated charges from the radical pair Pþ680Pheo�

on the acceptor side by electron transfer to QA in about 200 ps andto QB on the hundreds-of-ls time scale; and (e) on the donor side,an electron is supplied to reduce Pþ680 from a tyrosine residue (YZ)on the ns–ls time scale. It has been reported that the electrontransfer from YZ to Pþ680 is dependent on the ambient pH of thePS II preparations [11,12].

In contrast to the kinetics of electron transfer mechanisms,thermodynamic information is less accessible. Pulsed time-resolved photoacoustics provides a unique way to probe the ther-modynamic mechanism of electron transfer and proton transferevents in chemical reactions [13–19] and in photosyntheticcomplexes [20–29]. For a given photoreaction, the accessibleparameters include the molecular volume changes such as confor-mational change or electrostriction, enthalpy changes, and entropychanges. In addition, photoacoustics may provide information onkinetics and energy efficiencies in chemical systems andapplications of nanomaterials in solar energy storage [30–32].The analogous method of photothermal beam deflection BBD) alsoproduces similar data [33]. Thermodynamics reveals the energylevels of reactants and products, as well as the driving forces in achemical reaction. The driving force of a chemical reaction is theGibbs free energy, which is composed of enthalpic and entropiccomponents. The theory of electron transfer in chemical and bio-

Fig. 1. Structure and mechanism of PS II model of PS II (upper), Mn4Ca center (lower left), and S-state model (lower right) for oxygen evolution. Light-induced electrontransfer steps were labeled in red arrows. The valence of the Mn species in Mn4Ca cluster increased on S0 to S1 to S2 states, but less certain on S2 to S3 to S4 states. (Reproducedfrom [81]. with permission from the American Association for the Advancement of Science.)

Fig. 2. Pathways and rates of electron transfer reactions PS II.

358 H.J.M. Hou, D. Mauzerall / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 357–365

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logical system was developed by Marcus [34] and played a key rolein advancing the understanding electron transfer mechanisms,including photosynthetic systems [35–38]. However, a weaknessof the theory is the omission of entropic contribution in the theo-retical calculations of the free energy in the electron transfer steps[17,39–42].

There has been several comprehensive review articles summa-rizing the photoacoustic studies in the general aspect of photo-chemistry [15], photosynthesis [26,28,43,44], and PS I [29]. Inthis review we focus on the thermodynamics of charge separation,electron transfer, and oxygen evolution cycle in PS II, particularlyon the thermodynamic parameters including quantum yield, en-thalpy, entropy, and volume changes of PS II photochemistry. Thepotential limitations and future direction of thermodynamics ofPS II electron transfer and oxygen evolution are also presented.

2. Overview of photoacoustic measurements in photosynthesis

Photoacoustics is the ideal method to measure directly the vol-ume change (DV) and the enthalpy (DH) of a reaction by recordingand analyzing the sound wave caused by the volume change andthermal expansion or contraction of the process [15]. A micro-phone detects the photoacoustic waves via the thermal expansionin the gas phase [45–47]. A gas-coupled microphone in a closedchamber is used as a detector on the ms time scale. The 50-foldlarger thermal expansion of a gas over a liquid makes themicrophone sensitive. This method allows measurements of thephotosynthetic thermal efficiency, or energy storage, and of theoptical cross section of the light harvesting systems [48,49]. How-ever, the time resolution is low, typically about 30 ls, and thusinappropriate for obtain thermodynamic parameters on the fastertime scales. The use of piezoelectric films acoustically coupled toa liquid sample and a nanosecond pulsed laser light source in-creases the time resolution of the photoacoustic technique to themicrosecond and nanosecond scales [50,51]. Photoacoustic ther-modynamic studies have been carried out on isolated photosyn-thetic reaction centers from bacteria Rb. sphaeroides [52], on PS Ifrom cyanobacteria [53], on PS II using the PS II reaction centerD1/D2/cyt b559 complex from spinach [21], PS II core complexfrom Synechocystis [54], and the PS II complexes from spinachand from Chlamydomonas reinhardtii [53,55].

Measuring the energetics of photobiological reactions in vivo isof interest because the local environment of photoreceptors mayexert an important effect on their kinetics and thermodynamics.Pulsed photoacoustics via a microphone detector revealed the oxy-gen evolution and oxygen uptake in vivo on the millisecond timescale [46]. The uptake has been attributed to CO2 [56]. Using apiezo film detector, the in vivo enthalpy and volume changes ofphotosystem I and photosystem II of Synechocystis sp. PCC 6803were obtained on the microsecond time scale [57]. The contribu-tion of the two photosystems was distinguished by excitation attwo different wavelengths, 680 nm for predominant excitation ofPS I and 625 nm for PS II, respectively. A difficulty in the photoa-coustic measurements of intact cells is the heterogeneous or ‘‘cel-lular’’ artifact at the temperature of maximum density near 4 �C.The reason is that expansivity of the cellular matrix is not zero atthat temperature. Together with the finite size of the cell this intro-duces an artifact. To correct the ‘‘cellular’’ artifact, five measure-ments are needed instead of the usual three [57]. One mustmeasure the photoacoustic signals of the cell suspension at roomtemperature in the dark, at room temperature in background satu-rating light, at �4 �C in the dark and at �4 �C in saturating light aswell as the photoacoustic signal of a reference at room tempera-ture and combine these in a particular way. The enthalpy and vol-ume changes of intact cells of Synechocystis sp. PCC 6803 were in

good agreement with those of isolated complexes within theexperimental errors [29,57]. It is important to note that the PS IIcore centers are Mn depleted. The local protein environment ofYZ is undoubtedly changed and more open to the solution. This isprobably why the volume changes of PS II core complexes are sen-sitive to pH [54].

Theoretical calculations of electron transfer have often assumedthe reaction entropy to be zero. For example, the standard formu-lation of Marcus theory assumes that the vibrations coupled toelectron transfer have the same frequency in the reactant andproduct states [34]. Marcus theory introduces a reorganization en-ergy to interpret the reaction rate of electron transfer reactions.The reorganization energy can have two contributions: a vibrationterm and a solvent term. Treatments of the temperature depen-dence of the rate of electron transfer often assume that the freeenergy is independent of temperature [58]. However, theseassumptions are called into question by the observations of signif-icant reaction entropy in recent work [22,24,41]. The role of reac-tion entropy in the electron transfer reaction especially in proteinmay be substantial and cannot be neglected.

3. Thermodynamics of charge separation, electron transfer, andS-state cycle in PS II

3.1. Quantum yield

Quantum yield (U) of a photochemical reaction such as the lightinitiated charge separation in PS II is a fundamental parameter.This parameter is often assumed to be unity in photosynthesis.Using photoacoustics the quantum yield in photosynthesis can beobtained by analyzing the pulsed saturation curve [59,60]. For asimple system with one cross section, the photoacoustic signalfollows the cumulative one hit Poisson distribution. The fit of thisdistribution gives the effective cross section, which is the productof quantum yield and optical cross section (Ur). As the opticalcross section (r) with unit of area per reaction center can be calcu-lated from the in vivo absorption value, chlorophyll content, andthe number of centers in the sample, the quantum yield can bedetermined.

Fig. 3 shows the saturation curve of PS II photochemistry usingthe PS II core complex from Synechocystis sp. PCC 6803. Interest-ingly, the photoacoustic signal decreases dramatically when thepulsed energy is over 100–200 lJ/cm2, in the multi hit region.

1 10 1000

-2

-4

-6

-8

1

2

Vol

ume

cont

ract

ion

(A3 )

Pulse energy ( μJ cm-2)

Fig. 3. Saturation curves of PS II core complex at 3.7 �C using excitation at pH 6.0(closed circles) and at pH 9.0 (open circles). The lines are the calculated fits using asingle and double hit Poisson distribution equation. (Reproduced from [54] withpermission from the American Chemical Society.)

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The decrease in photoacoustic intensity may be due to a secondexcitation of the initial charge-separated state. The excitationmay cause charge neutralization and produces a positive volumechange. As the photoacoustic signal of the volume contraction viacharge separation in PS II is negative, the positive photoacousticsignal via charge neutralization would cancel part of the volumecontraction and give an overall decrease in photoacoustic intensity.The saturation curves calculated with a single and double hit Pois-son distribution equation and gave a good fit to the data (Fig. 3).The quantum yield of PS II charge separation of 100 ± 10% atpH 6.0 and pH 7.5 was observed. However the quantum yield isslightly lower, 83 ± 10%, at pH 9.0 [54].

As oxygenic photosynthesis involves two photosystems, thequantum yield of photosynthetic light reaction can be wavelengthdependent. The relative quantum yield spectrum of a reaction pro-vides useful information of the active pigments in photosynthesisand can easily be obtained by photoacoustics. The curve does notrepresent the absolute values but only relative values of photo-chemical activity as a function of wavelength of exciting light.The earlier photoacoustic quantum yield spectra of PS II in plantleaves using modulated light excitation showed the ‘‘red drop’’phenomena [61]. But there was an unexpected ‘‘red drop’’ in PS IIstarting from 670 nm instead of 680 nm and a drop in the Soretband region of chlorophyll a. This may be attributed to the closureof a noticeable fraction of PS II reaction centers under the steadystate conditions using modulated light sources. The intensity ofthe modulated light used to increases the signal to noise ratio issufficient to partially saturate the sample. Fig. 4 shows the quan-tum yield spectra of PS I and PS II from spinach. The quantum yieldat different wavelength indicates the efficiency of excitation en-ergy transfer between light harvesting and reaction center of thephotosynthetic systems. The ‘‘red drop’’ in PS II at 680 nm is evi-dent and expected. In contrast, PS I and overall PS I and PS II showan increase in the region absorbed by PS I [53,62]. This supportsour claim that more energy is stored by PS I than by PS II [63].

3.2. Molecular volume changes

The volume changes of charge separation and electron transferin PS II may be determined by pulsed photoacoustics at �4 �C. Atthis temperature, the volumetric thermal expansivity of water iszero. The heat component is zero, and the molecular volumechange is directly determined using the photoacoustic signals of

PS II and reference. As shown in Fig. 5, increasing the excitation en-ergy of the flash reduces the yield of the negative volume change.This is because multi-excitation of the ‘‘filled’’ reaction centers pro-duces no further charge separation. The maximum volume changeis the value measured at the lowest possible excitation energywhere the signal to noise raio is the poorest. By fitting the curveto the Poisson distribution one obtains the volume change at zeroexcitation energy with the least error.

Using the curve fitting methodology, the volume changes aredetermined at different pH values. These volume changes in PS IIcore preps for charge transfer are strongly pH dependent: �9 A3

at pH 6.0 and �3 A3 at pH 9.0. The electron transfer reaction fromYZ to Pþ680 occurs in >10 ls at pH 6.0 and <1 ls at pH 9.0, whenmeasured by absorption changes at room temperature in the UVand visible region [64–66]. Therefore the volume change of �9 A3

at pH 6.0 may be associated with the formation of Y�ZP680Q�A andpolar proton from the excited YZHP�680QA and -3 A3 may be pro-duced by the formation of Y�ZP680Q�A from the Y�Z P�680QA. Alterna-tively, the PS II centers might loss some of the QA during thepreparation. If this is the case, the smaller volume change maybe explained by the PS II centers with less QA.

The volume changes of PS II photochemistry obtained using PSII reaction center, PS II core complex, PS II oxygen evolving com-plex, whole cells, and PS I-less mutant are summarized and listedin Table 1. Upon excitation by a photon, the charge separation inPS II occurs to produce Pþ680Pheo� from the P�680. The volume changefor this charge separation reaction is small, ��2 A3 [21]. Thisseems small for a charge separation reaction in the protein andsuggests incomplete charge transfer in the QA depleted prepara-tion. In the complete system the electron in Pheo� is transferredto the secondary quinone acceptor QA. The volume changes forthe formation of Pþ680Q�A are in the range of �9 A3 to �13 A3

[26,54] smaller than seen in the bacterial center [52] Because onthe donor side, the Pþ680 receives an electron from Y�Z to formYZP680Q�A . The volume change for the step of formation ofY�ZP680Q A from Y�Z P�680Q A is estimated to be �1 A3 to �5 A3 becauseof charge transfer without charge separation.

The in vivo volume changes for charge transfer in the 0.1–10-lstime window in photosynthetic reaction centers of the intact cellsof Synechocystis PCC 6803 were determined using pulsed photo-acoustics. The volume change was determined to be �27 ± 3 Å3

and�2 ± 3 Å3 for PS I and PS II, respectively. The pulsed photoacou-stic method can correct the ‘‘cellular’’ or heterogeneous artifact and

Fig. 4. Quantum yield spectra of PS I and PS II from spinach by photoacoustics.(Reproduced from [53] with permission from Elsevier.)

2

1

1 10 1000

-1

-2

-3

-4

-5

-6

-7

-8

-9

-10

Vol

ume

yiel

d (A

3 /hν)

Pulse energy ( μJ cm-2)

Fig. 5. Volume yield of PS II core complexes versus light pulse energy at 3.7 �C atpH 6.0 (closed circles) and at pH 9.0 (open circles). (Reproduced from [54] withpermission from the American Chemical Society.)

360 H.J.M. Hou, D. Mauzerall / Journal of Photochemistry and Photobiology B: Biology 104 (2011) 357–365

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thus opens a route for studying the thermodynamics of electrontransfer on the microsecond time scale in vivo.

The unexpected observation is that the volume change (�9 to16 A3) of PS II preparations is significantly different from thein vivo data (�1 A3) [54,57]. One possible explanation is the finalstate is different in the two experimental conditions: Small D V’sare Y�ZP680Q�A from YZ

�P�680Q A in vivo. Alternatively, it may be dueto the different sample species: �1 A3 for cyanobacterium Synecho-cystis and �15 A3 for green algae and higher plants. This is unlikelygiven they are so similar. This discrepancy requires more work toconfirm or disapprove.

For the first time, the volume change for the S3 to S0 transitionin PS II S-state cycle is determined to be about +42 A3 (calculatedfor unity quantum yield) by PBD methodology [33,67]. The originof the large volume change may be the change of Mn’s nominalcharge from 2 of +IV to 2 of +II. Although it has been argued thatthe total charge on the OEC complex does not change during oxida-tion because of loss of protons this reduction step could cause alarge volume change because the electrostriction changes withthe square of the charge on the Mn ions. Thus, even if only partialcharge change occurs on the Mn’s, the volume change could beappreciable.

The volume contraction caused by electrostriction can be calcu-lated using the expended Drude–Nernst equation [68,69]:

DVel ¼@DGel

@P¼ e2 � j

2 � e

� �� @ ln e

@ ln V

� �� z2

þrþþ z2

�r�þ 2zþz�

r�

� �ð1Þ

where DVel is electrostriction, DGel is the Born charging energy, P ispressure, z+, z� are the charge on the positive and negative ions, j iscompressibility of the protein, V its molar volume, e its dielectriccoefficient or relative permeability, r+ and r� the radii of the donorand acceptor (assumed previously neutral) and r± is the distance be-tween the two ions. Note that this is a better approximation thanthe dipole formalism used by others, which contains unstatedassumptions.

PSII contains the donor tyrosine, YZ, D1-Tyr-161, which isdeprotonated before its oxidation by Pþ680 [70] possibly because ofthe effect of the electric field of Pþ680. The electron transfer fromY�Z to Pþ680 would cancel the charge of Pþ680 and Y�Z . The overall pro-cess would be the formation of Y�ZP680Q�A from Y�Z P�680QA, with nocharge separation, simply charge transfer. The difference betweenthe predicted value, +2 A3, and the observed �3 A3 could be causedby admixture with neutral histidine and oxidation of YZ with rapidtransfer of the proton resulting in the expected value of �30 A3.The data of PSII is best accommodated by a mixture of electrontransfer from anionic tyrosine and fast proton transfer to a polarregion dependent on pH [68].

As shown in Fig. 6, upon the photoinduced charge separation(step 1), at pH 6 the electric field of the chlorophyll cation inducesa fast proton transfer from protonated YZ to His near YZ (step 3),possibly D1-His-190. The proton is most likely released to the polarregion (step 4) then to the aqueous solution (step 5). At pH 9, elec-tron transfer from Y�Z to Pþ680 cancels the charges (step 2), leavingonly a charge movement, and thus gives a small volume changein the overall reaction.

3.3. Enthalpy changes

Using PS II core complex from Synechocystis sp. PCC 6803, theenthalpy change of PS II charge separation on the microsecondtime scale was determined by analyzing the photoacoustic signalsas in Fig. 7. Using the difference of slopes of d(PA�j)/da of PS II corereaction center and of ink reference, the enthalpy change in PS IIphotochemical reaction is �0.9 eV at pH 6.0, which is significantlarger than that (�0.43 eV) in PS I from the same cyanobacterium[59]. Thus more energy is stored in PS I than PS II on the microsec-ond time scale and also on the millisecond time scale [63]. The ob-served enthalpy change is pH dependent and increases from �0.9at pH 6.0 to �1.15 eV at pH 9.0. This is due to the participation ofYZ in the electron transfer in PS II at basic pH as discussed inSection 3.2.

The enthalpy changes, free energy, and entropy changes ofcharge separation, electron transfer, and S-state cycle in PS II aresummarized in Table 2. To determine the thermodynamic param-

Table 1Molecular volume changes of PS II photochemistry.

DV (A3) Reaction step Reference

PS II RC, spinach �2.5 ± 0.2 P�680Pheo! Pþ680Pheo� [21]PS II RC, spinach �0.5 ± 0.2 P�680Pheo! Pþ680Pheo� [21]PS II core, pH 6.0, Synechocystis �9 ± 1 Y�Z P�680QA ! Y�ZP680Q�A [54]PS II complex, spinach �9 ± 1 Y�Z P�680QA ! Y�ZP680Q�A [53]DEAE PS II complex, Chlamydomonas �11 Y�Z P�680QA ! Y�ZP680Q�A [26]BBY particles, spinach �16 Y�Z P�680QA ! Y�ZP680Q�A [26]PS II core, pH 7.5, Synechocystis �8 ± 1 Y�Z P�680QA ! Y�ZP680Q�A [54]PS II core, pH 9.0, Synechocystis �3 ± 0.3 HYZP�680QA ! BY�ZP680Q�A þ Hþ [54]DPsaB mutant (PS II), Synechocystisa �5 ± 0.5 HYZP�680QA ! BY�ZP680Q�A þ Hþ

Whole cells, Synechocystis �2 ± 3 Y�Z P�680QA ! Y�ZP680Q�A [57]PS II O2-evolving, spinach �13 Y�Z P�680QA ! Y�ZP680Q�A [33]PS II O2-evolving, spinach +40 S3 ? S0 [33,67]

a H.J.M. Hou and D. Mauzerall, unpublished data.

Fig. 6. Possible electron and proton transfer reactions in Mn-depleted PS II corecomplexes. Solid lines stands for the direction of electron transfer, and dashed linesfor proton transfer. D1/D2 are the reaction center core subunits. CP47 and CP43 arethe peripheral light harvesting proteins. (Adapted from [54] with permission fromthe American Chemical Society.)

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eters in the formation of Pþ680Pheo� from the P�680, PS II reaction cen-ter D1/D2/Cyt b559 complexes from spinach without QA were usedfor photoacoustic measurements. The enthalpy change accompa-nying formation of charge-separated state Pþ680Pheo� from theP�680 is �0.27 eV [21].

The in vivo photoacoustic measurements of whole cells of Syn-echocystis sp. PCC 6803 produced the enthalpies of �1.0 ± 0.2 eVfor the assumed formation of the ion radical pairs Y�ZP680Q�A [57].This value in vivo agrees well with the in vitro photoacoustic mea-surement of PS II core complexes (�0.9 to 1.1 eV) [54].

In addition, the thermodynamic parameters of oxygen evolutionin PS II were determined by PBD methodology [33]. The enthalpychanges for the formation of Y�ZP680Q�A from Y�Z P�680Q A is�0.82 ± 0.25 eV. Calculation of this value from the PBD data in-volved the assumption that the quantum yield of the PS II photo-

chemistry for excitation at 532 nm is 100%. It is roughly equal tothe estimated free energy �0.77 eV. The most interesting PBD datarevealed that the enthalpy change for the S3 to S0 transition is�0.2 ± 0.3 eV or maybe zero within the error range. The data ofMauzerall using air photoacoustics in leaves shows that the differ-ence in enthalpy of formation of the different S states is 0 ± 0.1 eV[71].

3.4. Entropy changes

The entropy of electron transfer reactions is often assumed tobe zero. However recent photoacoustic studies have shown thatsuch an assumption may not be true [22,24,41,72]. Using the trip-let state of zinc uroporphyrin in aqueous solution as electron donorand naphthoquinone-2-sulfonate as electron acceptor, it has beendemonstrated that the entropy (TDS) of this electron transfer reac-tion is quite large and negative, �0.6 eV [14]. The recent photoa-coustic data of bacterial reaction centers of Rb. sphaeroides [39]and PS I [41,59] revealed unexpected positive entropies, whichhave been attributed to bound counter ion release. Entropy changemay be correlated to Marcus reorganization energy [19]. However,a fit of Marcus reorganization energy cannot interpret the observedvolume change of electron transfer reaction [24]. Photoacousticsplays a key role in the determination of the entropy change of elec-tron transfer in proteins.

The enthalpy changes of a photoreaction are determined bypulsed photoacoustics (Table 2). Taking the free energy as the dif-ferences of their redox potentials in situ, the entropy change is cal-culated from DG = DH � TDS. We note that the in vivo DG’s arewrong for at least two reasons. (1) Redox measurements allow onlyone of the partners to be measured whereas the photochemicalreaction always has both the oxidized and the reduced form. Thishas unknown and possibly large effects on the proteins. The elec-trostatic effects alone may be 0.2 eV. (2) The redox measurementsare on the time scale of many minutes whereas those of thephotochemical reactions are ns to ls. Thus the protein environ-ment is not relaxed and the free energy is different. These entro-pies are ‘‘apparent’’ entropies and should be regarded as such. Asseen in Table 2 the entropy changes of charge separation and elec-tron transfer in PS II is generally small and negative, �0.1 to�0.2 eV within the errors, no matter what the pH of the solution.The small entropy change in PS II indicates that the PS II photo-chemistry is largely enthalpy driven. This is different from that inPS I, where some steps are entropy-driven [41]. Since the unex-pected positive entropy of the bacterial and PS I centers was as-signed to the liberation of counterions on charge formation, thisprocess does not occur in PS II centers since there may be no chargeformation on the ls time scale is Y�Z P�680QA goes to YZP680Q�A .

In the case of S-state cycle in PS II OEC, presumably each Sn toSn+1 transition includes proton and electron movement. Thesesteps are associated with the reorganization of hydrogen bondsat the Mn4Ca complex, proton release into the medium, Mn andYZ reduction by electrons from bound water, O–O bond formationand O2 release, reorganization of the Mn4Ca complex and the pro-tein matrix, and water binding. Each step of the process containsenthalpy and entropy changes. The PBD data give a very small en-thalpy change within the errors for the S3 to S0 transition and sug-gests an exergonic or an entropy-driven process (enthalpy � 0)[33]. Therefore the driving force of oxygen release and proton re-lease may be entropic.

3.5. Comparison of the thermodynamics of bacterial, PS I, and PS IIcenters

The volume changes (�28 Å3) for the formation of Pþ870Q�A in thebacterial reaction centers and that of PS I (�26 Å3) forming

Table 2Enthalpy changes, free energy, and entropy changes of charge separation, electrontransfer, and S-state cycle in PS II.

DH, eV DG, eV TDS, eV Reference

PS II RC, spinach �0.27 ± 0.1 �0.2 �0.1 [21]P�680Pheo! Pþ680Pheo�

PS II core, pH 6.0, Synechocystis �0.9 ± �0.1 �0.77 �0.1 [54]Y�Z P�680QA ! Y�ZP680Q�A

PS II complex, Synechocystis �0.58 ± 0.1 �0.77 +0.2 [54]Y�Z P�680QA ! Y�ZP680Q�A

PS II core, pH 9.0, Synechocystis �1.15 ± 0.2 �0.92 �0.2 [54]HYZP�680QA ! Y�ZP680Q�A þHþ

Whole cells, Synechocystis �1.0 ± 0.2 �0.77 �0.2 [57]HYZP�680QA ! Y�ZP680Q�A þHþ

PS II O2-evolving, spinach �0.3 to �0.8 �0.77 �0 [67]Y�Z P�680QA ! Y�ZP680Q�A

PS II O2-evolving, spinach �0 ± 0.3 [67]S3 ? S0 transition

-50 0 50 100 150 200 250

-200

-100

0

100

200

300

3

2

1

κ PA

α 106 (oC-1)

Fig. 7. The product of photoacoustic signal and water compressibility (j) versuswater thermal expansivity (a) for carbon ink reference and PS II core complexes.The open circle symbols (line 1) are the reference. The closed square symbols (line2) represent the measurement at pH 9.0, and the closed circles (line 3) are obtainedat pH 6.0. The lines are the linear fit of the experimental data. (Reproduced from[54] with permission from the American Chemical Society.)

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Pþ700FA=F�B are quite similar [39,59]. A smaller volume contraction,about �9 Å3 for the formation of Y�ZPþ680Q�A with polar proton andabout �3 Å3 for Y�ZP680Q�A in PS II is measured. The former datacan be explained by electrostriction but assuming similar dielectricand thermoelastic properties, the data on PS II requires that if thedonor is YZH it must be linked to rapid proton expulsion. If Y�Z is thedonor, the small volume change is simply explained. If protontransfer to polar environment occurs it must occur in <100 ns.The pH dependence of the PS II core preparations supports thisview.

The enthalpies of electron transfer in bacterial and PS I reactioncenters were found to be �0.44 eV and �0.35 eV, respectively[39,59]. The similar difference in redox potentials of the donorand acceptor produce similar apparent entropies of reaction. How-ever, the photoacoustic data indicate that the enthalpy change ofphotochemistry on the 1-ls time scale in PS II is larger (�1 eV)than that of PS I and bacterial reaction centers (�0.4 eV). Theapparent entropy of electron transfer in PS II is negative, which isnormal for charge formation in solution [14,40]. The electrontransfer reaction in PS II is associated with proton transfer, whichis favored by a more polar environment in the complex. Overall,the thermodynamic data of bacterial, PS I and PS II reaction centersshow that the entropy of electron transfer is complex and dependsupon the microenvironment of the protein and may involve cou-pling with proton transfer in PS II. This complexity of electronand electron/proton transfer reactions requires detailed and sys-tematic studies using pulsed photoacoustics in order to understandthe mechanism of electron transfer in photosynthetic systems.

If the hypothesis of proton transfer in PS II is correct, it must oc-cur in <100 ns, too fast to be time-resolved using ls pulsed photo-acoustics. We have developed a fast pulsed, time-resolvedphotoacoustics setup with time resolution of <10 ns. Experimentswith this cell may allow one to time-resolve the components ofthe YZH! Pþ680 reaction which we would predict to have largeand opposite volume changes. Another test would be YZ-lessmutant (e.g. D1-Tyr161Phe [73]) PS II core complexes, which isexpected to form Pþ680Q�A upon photoinduced charge separationon ls time scale. This preparation would enable one to measurethe volume contraction and enthalpy producing the Pþ680Q�A fromexcited P�680 without interruption of electron/proton transfer stepfrom YZ.

4. Limitations and potential problems

Some of the thermodynamic parameters of charge separation,electron transfer, and the S-state cycle in PS II complexes from cya-nobacteria, green algae, and higher plants, and in whole cells ofSynechocystis have been determined using pulsed photoacoustics[54,57,59,67]. However, the parameters in Synechocystis PS II wereinconsistent with those obtained using Chlamydomonas and spin-ach chloroplast [53,74]). The in vitro measurements, in particular,showed a significant difference from the in vivo values. These dis-crepancies in purified complexes from different organisms such asSynechocystis, Chlamydomonas, and spinach chloroplasts must beclarified.

In contrast to the volume changes in PS I and bacterial reactioncenters, which agree within the error, the volume change of PS IIcharge separation is in the range from �2 to �16 Å3 [26]. Thisinconsistency needs to be investigated and clarified. The differentpH, species of organisms, preparations, redox potentials of electrondonor/acceptors, and particular the cellular artifacts in the photoa-coustic measurements may cause the discrepancy.

The volume change of our previous photoacoustic measure-ments showed a strong pH dependence of volume change in PS IIcore complexes. A volume change of �3 Å3 was observed at

pH 9.0 and �9 Å3 at pH 6.0 [54]. Future work may determine thevolume change of charge separation in PS II complexes using ourprevious photoacoustic procedures over a range of pH. The volumechange will be measured by the yield and saturation method,respectively. As Delosme et al. pointed out, the discrepancy ofthe volume change may be due to the use of different organisms:cyanobacteria versus green plants [26]. The PS II preparationscan be purified from cyanobacterium Synechocystis 6803, fromgreen algae Chlamydomonas reinhardii, and from spinach chloro-plasts following established methods [75–78]. The volume changeof charge separation in PS II should be determined and evaluatedagainst the data from different groups.

The inconsistency in volume changes and enthalpies of electrontransfer could be due to the difference in intactness: in vitro versusin vivo. Most of the in vitro preparations gave around �10 Å3 com-pared to �2 Å3 for the in vivo system. A number of factors in vivoare different from the in vitro preparations. Use of the PS I-depletedmutant may be required to address this problem. Based on preli-minary unpublished data Delosme et al. argued that the thermalefficiency of purified PS II core from Thermosynechococcus elongatusstrongly depends on the experimental conditions [26], such as theartificial electron donor or acceptor used, and could be significantlyhigher than our previous data. This discrepancy needs to beclarified.

It has been a long-term goal to reveal the thermodynamics of PSII oxygen evolution. Recently, using photothermal beam deflectiontechniques some of the enthalpies of PS II water oxidation were re-ported [33]. However, the PBD data is obtained at 532 nm and in-volves significant heat loss. The experimental errors are ratherlarge. Further improvement in the measurement of PBD methodol-ogy is needed. The main problem for pulsed photoacoustic to as-sess the PS II water oxidation is the time window, which is onthe microsecond time scale. Therefore it is too fast to determinethe millisecond step of PS II oxygen evolution cycle. Using an airmicrophone and leaves Mauzerall has obtained enthalpy changesof the S state formation [71]. In addition, a novel photopressure cellthat enabled one to obtain the volume and enthalpy changes of thephotocycle in bacteriorhodopsins on the microsecond to secondtime scales [79,80], may allow the thermodynamic parameter ofreactions involved in PS II oxygen evolution to be determined.

5. Conclusions

Knowledge of both the thermodynamics and the kinetics of areaction are required for its detailed understanding. However, incontrast to the kinetics of electron transfer in PS II, thermodynamicinformation is still limited. In particular, the discrepancies of thereported data and the unexpected experimental results in PS IIthermodynamics need to be clarified and explained. Pulsedphotoacoustic methodology enables one to extract directly ther-modynamic parameters, which include the molecular volumechange via conformational change or electrostriction and energylevels of charge separation, electron transfer, and S-state cycle inPS II. Recent results show that some electron transport steps areenthalpy-driven in PS II electron transfer. Surprisingly, the S-statecycle in PS II OCE is likely entropy-driven [67] as are steps in PS I[22,41]. Further work on these interesting systems is especiallyworthwhile.

Recent progress in thermodynamic measurements provides no-vel insights on the enthalpic and entropic contribution to electrontransfer in proteins and is able to probe the role of the membraneenvironment and cofactors in vitro and in vivo in terms of a broadlythermodynamic view [60]. These unique features provide the po-tential for a deeper understanding of the mechanisms of electrontransfer and proton-coupled electron transfer in chemical and bio-

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logical systems. They also provide a framework to modify and im-prove the existing Marcus electron transfer theory as well as theformulation of a comprehensive electron transfer theory. The re-sults also provide direct information about the function of mem-brane proteins central to photosynthesis. In particular, thephotosynthetic membrane proteins are excellent models for eluci-dating electron transfer and proton transfer mechanism in othermembrane proteins. The thermodynamic knowledge discussedhere may have profound impact on the bioenergetics of electrontransfer mechanisms in chemistry and biology.

Acknowledgments

The work was supported by previous grants from the NationalScience Foundation (MCB-9904522 to D.M.) and by University ofMassachusetts Dartmouth (to H.J.M.H.). The authors are gratefulto Vladimir A. Boichenko and Bruce A. Diner for productive collab-oration on PS II, Gregory Edens for useful discussions, and IreneZielinski-Large for technical assistance. They thank Fan Zhang, ZiWei, Lien-Yang Chou, Xuejing Hou, and Wanshu He for helpfuldiscussions.

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