Proc. Natl. Acad. Sci. USAVol. 90, pp. 10942-10946, December 1993Biochemistry

A functional model for the role of cytochrome b559 in the protectionagainst donor and acceptor side photoinhibition

(functional model)

JAMES BARBER AND JAVIER DE LAS RIVAS*Agricultural and Food Research Council Photosynthesis Research Group, Wolfson Laboratories, Department of Biochemistry, Imperial Coliege of Science,Technology and Medicine, London SW7 2AY, United Kingdom

Communicated by Daniel I. Arnon, July 6, 1993

ABSTRACT A quinone-independent photoreduction of thelow potential form of cytochrome b55, has been studied usingisolated reaction centers of photosystem II. Under anaerobicconditions, the cytochrome can be fully reduced by exposure tostrong illumination without the addition of any redox media-tors. Under high light conditions, the extent and rate of thereduction is unaffected by addition of the exogenous electrondonor Mn2+ and, during this process, no irreversible damageoccurs to the reaction center. However, prolonged mluminationin strong light brings about irreversible bleaching of chloro-phyll, indicative of photoinhibitory damage. When the cy-tochrome is fully reduced and excess Mn2+ is present, the effectof moderate light is to facilitate the photoaccumulation ofreduced pheophytin. The dark reoxidation of the reducedcytochrome is very slow under anaerobic conditions but sig-nificantly speeded up on addition of oxidized 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. From these results it issuggested that the low potential form of cytochrome b5sg canaccept electrons directly from reduced pheophytin and in sodoing help to protect the reaction center against acceptor sidephotoinhibition as suggested by Nedbal et al. [Nedbal, J.,Samson, G. & Whitmarsh, J. (1992)Proc. Natl. Acad. Sci. USA89, 7929-7933]. This conclusion has been incorporated into amodel that further suggests that in its high potential form thecytochrome primarily acts to protect against donor side pho-toinhibition due to increased lifetime of highly oxidized speciesas previously proposed by Thompson and Brudvig [Thompson,L. & Brudvig, G. W. (1988) Biochemistry 27, 6653-6658]. Theparticular feature of our scheme is that it incorporates revers-ible interconversion between the two redox forms so as toprotect against either type of photoinhibition.

Cytochrome b559 (cyt b559) is closely associated with thereaction center of photosystem II (PSII) as judged fromkinetic (1, 2), structural (3-5), and molecular biological (6)studies. Ofthe many different suggestions for its function, themost popular is that it helps to protect PSII against photo-induced damage (7). In this paper we show that cyt b559 canbe directly reduced by pheophytin (Pheo) and propose ascheme by which cyt b559 could prevent photoinhibitorydamage of PSII due either to "acceptor" or "donor" sideevents (8). The proposed scheme requires cyt b559 to bereversibly convertible between its high-potential (Em +400mV) and low-potential (E = +60-80 mV) forms.An understanding of the distinction between donor and

acceptor side photoinhibition is relatively new (8). Donor sidephotoinhibition occurs when the rate of electrons leaving thePSII reaction center is greater than the rate of donation.Consequently the lifetime of the primary electron donorP680+, and other secondary oxidized species, increases. Thedamage incurred by this effect is thought to be due to the high

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oxidizing potentials of long-lived species (7, 8). Donor sidephotoinhibition is readily seen when the water-splitting re-actions have been inhibited (9-11). Acceptor side photoin-hibition occurs when there is an overreduction ofthe quinonepool and QA becomes doubly reduced (12, 13). In this event,there is an increase in the probability ofcharge recombinationbetween the primary radical pair P680+ Pheo- leading to thetriplet state of P680. This triplet state can interact withoxygen and form highly reactive singlet oxygen.

Support for these mechanisms comes mainly from studiesusing in vitro systems. A particularly useful experimentalsystem has been the isolated reaction center of PSII. Thiscomplex consists of the Dl and D2 proteins (products of thepsbA and psbD genes, respectively), the a and p apoproteinsof cyt b559, and the product of the psbl gene (3, 14). Thisisolated reaction center does not contain the secondaryquinone electron acceptors QA and QB and therefore isrestricted in its photochemical activities to radical pair for-mation and recombination (15-17). Addition of exogenouselectron donors and acceptors, however, allows secondaryelectron flow reactions to occur (14). Of relevance to theideas presented in this present paper was the finding that bothacceptor and donor side photoinhibition can occur in thisrelatively simple system. When no additions are made, theacceptor side mechanism occurs since singlet oxygen isformed as a consequence of radical pair recombination (18).The generation of this highly toxic species causes initially aselective and irreversible bleaching of the chlorophylls thatconstitute P680 (ref. 19) and a breakdown of the Dl proteinto a 23-kDa fragment containing the N terminus of thecomplete protein (20). This fragment is possibly the same asthat observed in vivo by Greenberg et al. (21) and seems tobe due to a proteolytic cleavage in the loop joining theputative transmembrane helical segments four and five in theregion of the QB binding site. If, however, an electronacceptor is present, the P680+ lifetime is increased, andirreversible bleaching of carotenoid and chlorophyll occurs,which is independent of oxygen (19). Under these conditionsthe donor side mechanism prevails, and breakdown of the Dlprotein leads to a 24-kDa fragment of C-terminal origin (22,23). The cleavage site in this case is likely to be on the lumenalside of the complex, probably in the loop joining transmem-brane segments one and two. Similar conclusions have beendrawn from experiments utilizing isolated oxygen-evolvingPSII cores (24).There are many reports showing that the high-potential

form of cyt b559 (cyt b559Hp) can be converted to a low-potential form of cyt b559 (cyt b559Lp) (25). This conversion is

Abbreviations: cyt b559, cytochrome b559; cyt b559Hp and cyt b559Lp,high- and low-potential forms of cyt b559; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; Pheo, pheophytin; PSII, pho-tosystem II.*Present address: Department of Biochemistry, Faculty of Science,University of the Basque Country, P.O. Box 644, Bilbao, Spain.

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Proc. Natl. Acad. Sci. USA 90 (1993) 10943

readily observed when PSII particles are isolated by usingdetergents (e.g., ref. 26). But the conversion also occurswhen isolated membranes are subjected to various treat-ments not involving detergents, such as exposure to photo-inhibitory light (27) or removal of the extrinsic proteins ofthewater-splitting complex (e.g., ref. 28). Importantly it has beenshown that cyt b559Lp can be converted back to cyt b559Hp by,for example, the addition of certain lipids (29) or rebinding ofextrinsic PSII proteins (30). There is also good evidence thatcyt b559Hp and cyt b559Lp exist in vivo, and indeed theinterconversion of the two redox states has formed the basisof functional models proposed to explain the water-splittingreaction (31) or in relation to a redox link between PSII andPSI (32).

Within the isolated reaction center, cyt b559 exists mainlyin its low potential form. Because the ambient redox potentialis usually in the region of 100-150 mV, the cytochromenormally exists in its oxidized state. However, in the pres-ence of added quinones and under aerobic conditions, it wasshown that it could be photoreduced (33, 34). In this paper weshow that cyt bs59Lp within the isolated reaction center can bedirectly reduced by Pheo and present a model for the role ofcyt b559 as a protectant for both acceptor and donor sidephotoinhibition.

MATERIALS AND METHODSThe reaction center of PSII was isolated from pea (Pisumsativum) according to the method given in Chapman et al.(33). The chlorophyll concentration of the reaction centersolution used was 4 pg/ml, calculated by the method ofArnon (35). The samples were suspended in 50 mM Mes-NaOH (pH 6.0) containing 2 mM dodecyl maltoside and 0.2M sucrose. For preillumination treatments, the samples weremaintained at 10°C with a thermally controlled cuvetteholder. Anaerobic conditions in the samples were obtainedby adding glucose oxidase (0.1 mg/ml) and catalase (0.1mg/ml) plus 5 mM glucose and incubating for 5 min beforemeasurements or by extensive degassing of all the solutionswith oxygen-free nitrogen and sealing of the sample cuvettein a stream of nitrogen.

,4min-2 min--1 min

-0.5 min5

x

0

l0

03

Absorption spectra were measured at 10°C in a dual beamspectrophotometer (Aminco, SLM Instrument; model no.DW2000) set with a bandwidth of 2 nm. Where indicated, thereaction center solutions were preilluminated with heat-filtered white light with intensities and times as described inthe figure legends. Photoinduced absorbance changes atdifferent wavelengths (559, 450, and 422 nm) were measuredwith the same spectrophotometer by using side excitation ofa 1-ml, 1-cm path length sample cuvette with heat-filtered redlight (RG660 filter) and with the photomultiplier shielded bya complementary blue filter (Coming 4-96). Light-minus-darkdifference spectra were obtained by automatic scanning aftergiving a particular exposure to illumination.

Quantification of the level of cyt b559 in reaction centersamples was carried out by absorption spectroscopy after theaddition of 1-2 mM ferricyanide in the dark, followed by theaddition ofa few grains of sodium dithionite (-2 mg/ml). Thedithionite-minus-ferricyanide difference spectrum, from 600to 500 nm, was obtained, and the AA was measured at 559 nm.

RESULTSFig. 1 shows the absorption spectra of isolated reactioncenters that had been preilluminated with white light minusthe dark control. The light-minus-dark difference spectrawere recorded at various times during the illumination periodand are characterized by two main positive bands at 430 and559 nm that are typical of cyt b559 reduction. The differencesin the spectra shown in Fig. 1 are due to the presence ofMn2+, which, in particular, distorted the AA at about 410 nm.The kinetics of the photoreduction of cyt b559 followed amonoexponential curve with a rate constant estimated to be0.08 sec-1. It was also found that there was no significantdifference between the reduction rates in the presence orabsence of Mn2+ when strong intensity illumination wasused. This indicates that under anaerobic conditions thereaction center can provide an electron to reduce the cy-tochrome without being damaged (note that in Fig. 1 nophotobleaching of pigments was observed, suggesting thatthe electron donor was neither chlorophyll nor 1-carotene).

400 500 600 400 500 600Wavelength, nm

FIG. 1. Absorption difference spectra of the illuminated reaction centers minus the dark control. Illumination was with white light [800,uE*m-2.s-1; 1 einstein (E) = 1 mol of photons] for 0.5, 1, 2, and 4 min, in the presence of 0.5 mM MnCl2 as electron donor (A) and in absenceof MnCl2 (B). The experiments were carried out under anaerobic conditions using a glucose/glucose oxidase trap (see Materials and Methods).The chlorophyll concentration was 4 Ag/ml.

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x

.00

-1 4 min,,"~~~~~~~~ 5minKF 1 680

500 600 700Wavelength, nm

FIG. 2. Absorption difference spectra of the illuminated reactioncenters minus the dark control. Illumination was with white light(1500 ,E.m-2 s-1) for 2, 3, 4, and 5 min in the presence of 0.5 mMMnCl2 as electron donor and in anaerobic conditions. The chloro-phyll concentration of the light-treated samples was 4 pg/ml.

A possible donor could be an amino acid residue such astyrosine or histidine (36-39).A comparison of the maximum extent of the light-induced

reduction of cyt b559 with its chemically induced reduction inthe dark (dithionite-minus-ferricyanide) gave practically thesame AA559. The absorbance change corresponded to thereduction of 1 mol of cyt b559 for every 6.5 mol ofchlorophyllwhen the e at 559 nm was taken as 17.5 mM-1 cm-1 (40).Considering that every reaction center contains one cy-tochrome and six chlorophyll molecules (41, 42), the resultsabove indicate that the photoaccumulation of reduced cytb559 occurs in nearly all the reaction centers (i.e., 93% of thecenters).

Fig. 2 shows the irreversible pigment bleaching that occurswhen the isolated reaction centers are subjected to strongillumination for long periods of time (2-5 min). After 2 min,all the cyt b559 was reduced and only then did furtherillumination provoke bleaching in the red region, peaking at680 nm, indicating irreversible damage of P680 and otherchlorophylls (19). This therefore indicates a possible protec-tive role of cyt b559 acting as an electron acceptor from Pheo

422 nm 450 nnr

559nm

x

U.0 ~ ~ ~ ~ ~ J~~A A

AA 450nm ( 450nmm

Time, min

and thus decreasing the probability of damage by radical pairrecombination (19).The photochemical activity of samples in which reduced

cyt b559 had been photoaccumulated by exposure to actiniclight under anaerobic conditions was measured. First, in darkpretreated samples, the actinic light induced the reduction ofcytochrome, indicated by an increase of AA at 559 nm (Fig.3A). In these samples no chromatic change was detectedwhen monitored at 450 nm. After a 5-min illumination, all thecyt b559 was reduced, and then, in these light-treated samples,we observed the reversible photoinduced reduction of Pheo(Fig. 3B). The anion radical of Pheo (Pheo-) is spectrallycharacterized in Fig. 3B by the reversible light-inducedpositive signals at 450 nm and negative at 422 nm (3, 43) andby the light-minus-dark difference spectrum presented in Fig.3C. This difference spectrum is similar to that reportedpreviously (14) and is not distorted by irreversible bleachingof chlorophyll since the intensity of the actinic light wassignificantly lower than that used to obtain the data of Fig. 2and also because only the reversible difference spectrum hasbeen recorded. This result indicates that when all the cyt b559is reduced in the reaction center, a reversible photoaccumu-lation of reduced Pheo can occur. This photoinduced reduc-tion of Pheo was maximum in the presence of Mn2+ but didoccur to a lesser extent without added electron donor. Thequantification of this optical change observed at 450 nm andmeasured with excess Mn2+ present indicated that the pho-toaccumulation of reduced Pheo occurs in 80% of the reac-tion centers [considering that one Pheo is reduced in eachreaction center and using the e at 450 nm of 8.3 mM-1'cm-1,calculated from Nanba and Satoh (3)].The data presented so far imply that the photoreduction of

cyt b559 is irreversible, since the difference spectra shown inFig. 1 were stable in the dark, at least over a period longerthan 10 min. If, however, 100 ,M oxidized 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) was intro-duced into the anaerobic suspension, there was rapid reox-idation of the photoaccumulated reduced cytochrome (seeFig. 4). Indeed, when 100 ,uM DBMIB is present during thepreillumination period, no accumulation of reduced cyt b559is observed.

DISCUSSIONThe discovery by Knaffand Arnon (1) that cyt b559Hp could bephotooxidized at low temperatures when the water-splittingreactions were inactivated was the first direct evidence that

FIG. 3. Kinetics of the light-inducedabsorbance changes at 450 and 559 nm ofthe isolated reaction centers before preil-lumination (A) and after S min of illumi-nation with 800 uE-m-2.s- white light(B). (C) Automatically recorded light-

545 nm minus-dark difference spectrum of thereversible changes due to the photore-duction of Pheo. In all cases MnCl2 (0.5mM) was present, and anaerobic condi-tions were achieved with a glucose/glucose oxidase trap. The chlorophyll

C concentration was 4 ,Ag/ml. Excitationwas with 100 tE m-2s-1 red light. LightI on, upward open arrowhead; light off,

500 600 downward filled arrowhead. The bar in--length, am dicates an absorbance change of 10-3.

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xa)

co0.0

500 600Wavelength, nm

700

FIG. 4. The effect of introducing 100 ,uM DBMIB into an anaer-obic sample of PSII reaction centers that had been preilluminated for5 min with 800 j,E'm-2.s-' white light so as to fully reduce cyt b559.Spectrum a, light-minus-dark difference spectrum before addingDBMIB; spectrum b, spectrum after the addition of quinone.

this component is closely associated with the reaction centerof PSII. Their work was confirmed by others (2) and formedthe basis of a host of different postulates to describe thefunctional role of this heme in PSII activity. Isolation of thePSII reaction center (3, 14) confirmed that the cytochrome isintimately associated with the D1/D2 heterodimer and em-phasized a significant difference in the composition of thehigher plant system as compared with the reaction center ofpurple photosynthetic bacteria. Another striking differencebetween the reaction centers ofPSII and purple bacteria is thehigh vulnerability ofthe former to photoinhibitory damage andthe remarkably high turnover rate for the Dl protein (8). Thus,of the many suggestions as to the function of cyt b559 withinPSII, the most popular has implicated a protective role againstphotoinhibition (e.g., ref. 7).Our understanding of the molecular processes of photoin-

hibition have advanced considerably in recent years. Much ofthis advancement has come from studies with in vitro systemsranging from isolated thylakoids (9, 10, 27) and PSII-enrichedmembrane fragments (11, 12) to isolated oxygen-evolvingPSII complexes (24) and PSII reaction centers (20, 22, 23). It

now seems clear that both donor and acceptor side photoin-hibition can occur (8). Recognition of this fact thereforeopens up the question of how cyt b559 could protect eitherroute of photoinduced damage. Thompson and Brudvig (7)have presented a detailed scheme of how cyt b559 could serveas a protectant against donor side photoinhibition. In thisscheme, cyt b559Hp is redox poised so as to supply an electrondirectly or indirectly to P680+ when electron flow from wateroxidation is inadequate. More recently, Nedbal et al. (44)have, from indirect measurements, implied that cyt b559Lp canact as a direct oxidant of reduced Pheo and in so doing,protect PSII against acceptor side photoinhibition.The experiments presented in this paper clearly demon-

strate that the light-driven photoreduction of cyt b559 canoccur as a consequence of direct electron transfer fromreduced Pheo. Both the photoaccumulation of Pheo-, whenall the cytochrome is reduced, and the lack of any quinone inthe isolated reaction center complex support the conclusionthat the electron donor to cyt b559 is Pheo. Quantification ofthe signal at 559 nm indicates that the cytochrome can bereduced in all the reaction centers. We estimate the quantumyield of this light-induced reduction to be about 0.025 (2.5%)based on 5% absorption of incident light and 100%o efficiencyfor primary charge separation.Our results give direct experimental support to the hypoth-

esis of Nedbal et al. (44)-that is, the cyt b559Lp can acceptelectrons from reduced Pheo reasonably efficiently and thusreduce the risk of damage caused by radical pair recombi-nation (8). The other important result of our work is that thereduced cytochrome is not efficiently reoxidized by the donorside of PSII as would be expected if an effective cyclicprocess were operative. Nevertheless, such a cycle hasfrequently been suggested. To explain our observations wewish to propose a model that recognizes that cyt b559 can existin a high and low potential form and that both donor andacceptor side photoinhibitory processes occur. We also as-sume that there is only one cyt b559 per functional PSII in thethylakaid membrane for which there is now very strongexperimental evidence (41, 45). Our model is schematicallyshown in Fig. 5 in which we propose a reversible changebetween high and low potential forms of cyt b559 with thepurpose of protecting against either donor or acceptor sidephotoinhibition. In its high potential form (Em +400 mV),cyt b559 is maintained in its reduced state and therefore ideally

v Acceptor sideXImpalrment

(A

\QBCytb5Cyt 59LP

Ambient T I epAotentat molecular switch

Cytb559HP

Water

P6880Donor sideImpairment

FIG. 5. A scheme to emphasize the possible role ofcyt b559 as a one-electron protectant against acceptor ordonor side photoinhibition. The model postulates thatin order for the cyt b559 to act either as an electronacceptor (for acceptor side protection) or as an electrondonor (for donor side protection) it shifts reversiblybetween its high and low potential forms, triggered byan unknown molecular switch. The reoxidation ofreduced cyt b559Lp and the reduction of oxidized cytb559Hp is suggested to involve electron exchange withthe ambient redox system governed by factors such asthe redox state of the plastoquinone pool and thepresence of molecular oxygen.

- 0.5V-

ov .

+O.5V-

+ l.OV'

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10946 Biochemistry: Barber and De Las Rivas

poised to give an electron to long lived, and potentiallydangerous, oxidized species on the donor side of PSII. Onceoxidized, the cytochrome can be rereduced by the ambientpotential of its surroundings governed, for example, by theredox state of the plastoquinone pool. The slowness of therereduction of photooxidized cyt b559 has been noted previ-ously (46). However, under conditions when acceptor sidephotoinhibition might occur due to complete reduction ofQAand QB (12, 13), cyt b559 could shift its potential to the lowpotential form. Such a shift in response to strong illuminationhas already been observed (27) and was faster than thesubsequent appearance of photoinhibitory damage. In its lowpotential form, cyt b559 exists in an oxidized state andtherefore is ideally poised to act as an electron acceptor. Thereoxidation of the reduced cyt b559Lp would occur due to theambient potential of the system, which again could be gov-erned by the redox state of the plastoquinone pool. Indeedthe experiment presented in Fig. 4 indicates the effectivenessof DBMIB in oxidizing the photoreduced heme within theisolated PSII reaction center.The scheme shown in Fig. 5 suggests that the conversion

between cyt b559Hp and cyt b559Lp is reversible. Although theconversion from high to low potential is well documented, thereverse is less commonly observed. However, such a con-version does occur in vivo when non-QB PSII complexes,located in stromal lamellae, are "activated" to becomeoxygen-evolving QB active PSII complexes in the grana (47).The nature of the "molecular switch" that allows this inter-conversion to occur is unknown but is likely to involve achange in the environment of the heme or a minor confor-mational change of the PSII complex as indicated from invitro studies (25-28). One obvious mechanism could involveprotonation/deprotonation events associated with changes inlocal pH. Although the proposed model needs further exper-imental support, we hope that it will give a new frameworkon which to discuss the functional role of cyt b559 in photo-synthesis. In its final form it will need to reconcile theexistence of data that, at first sight, do not easily fit into thismodel, including the work of Buser et al. (48, 49), whichimplicates the QB site in the photoreduction of cyt b559.

We acknowledge the technical support of Caroline Woollin. Wewish to thank the Agricultural and Food Research Council, TheFederation of Biochemical Societies, and the Spanish governmentfor financial support.

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