Biochimica et Biophysica Acta

Transcription

1 Biohimi et Biophysi t 1807 (2011) Contents lists ville t SieneDiret Biohimi et Biophysi t journl homepge: Proing the quinone inding site of Photosystem II from Thermosynehoous elongtus ontining either Ps1 or Ps3 s the D1 protein through the inding hrteristis of heriides lin Bouss,, Miw Sugiur,, Frie Rppport, ibite-s, CNRS UR 2096, CE Sly, Gif-sur-Yvette, Frne Cell-Free Siene nd Tehnology Reserh Center, Ehime University, Bunkyo-ho, Mtsuym Ehime, nd PRESTO, JST, Honho, Kwguhi, Sitm, , Jpn Institut de Biologie Physio-Chimique, UMR 7141 CNRS nd Université Pierre et Mrie Curie, 13 rue Pierre et Mrie Curie, Pris, Frne rtile info strt rtile history: Reeived 15 July 2010 Reeived in revised form 23 Septemer 2010 epted 4 Otoer 2010 ville online 16 Otoer 2010 Keywords: Photosystem II D1 protein Ps protein Heriide EPR sorption hnge The min oftors involved in Photosystem II (PSII) oxygen evolution tivity re orne y two proteins, D1 (Ps) nd D2 (PsD). In Thermosynehoous elongtus, thermophili ynoterium, the D1 protein is predominntly enoded y either the ps 1 or the ps 3 gene, the expression of whih depends on the environmentl onditions. In this work, the Q B site properties in Ps1-PSII nd Ps3-PSII were proed through the inding properties of DCMU, ure-type heriide, nd romoxynil, phenoli-type heriide. This ws done y using helium temperture EPR spetrosopy nd y monitoring the time-resolved hnges of the redox stte of y sorption spetrosopy in PSII purified from His 6 -tgged WT strin expressing Ps1 or from His 6 -tgged strin in whih oth the ps 1 nd ps 2 genes hve een deleted nd whih therefore only express Ps3. It is shown tht, in oth Ps1-PSII nd Ps3-PSII, romoxynil does not ind to PSII when Q B is in its semiquinone stte whih indites muh lower ffinity for PSII when is in its semiquinone stte thn when it is in its oxidized stte. This is onsistent with the midpoint potentil of Q / eing more negtive in the presene of romoxynil thn in its sene [Krieger-Liszky nd Rutherford, Biohemistry 37 (1998) ]. The ddition in the drk of DCMU, ut not tht of romoxynil, to PSII with seondry eletron eptor in the Q B stte indues the oxidtion of the non-heme iron in frtion of Ps3- PSII ut not in Ps1-PSII. These results re explined s follows: i) romoxynil hs lower ffinity for PSII with the non-heme iron oxidized thn DCMU therefore, ii) the midpoint potentil of the Fe II /Fe III ouple is lower with DCMU ound thn with romoxynil ound in Ps3-PSII; nd iii) the midpoint potentil of the Fe II /Fe III ouple is higher in Ps1-PSII thn in Ps3-PSII. The oservtion of DCMU-indued oxidtion of the non-heme iron leds us to propose tht Q 2, n eletron eptor identified y Joliot nd Joliot [FEBS Lett. 134 (1981) ], is the non-heme iron Elsevier B.V. ll rights reserved. 1. Introdution Light-driven wter oxidtion y the Photosystem II (PSII) enzyme is responsile for the prodution of O 2 on Erth nd is t the origin of the synthesis of most of the iomss. Refined three dimensionl X-ry strutures from 3.5 Å to 2.9 Å resolution hve een otined using PSII revitions: PSII, Photosystem II; Chl, hlorophyll; PPBQ, phenyl-p-enzoquinone; MES, 2-(N-morpholino) ethnesulfoni id; P 680, primry eletron donor;,primry quinone eptor; Q B, seondry quinone eptor; TL, thermoluminesene; 43H, T. elongtus strin with His-tg on the C terminus of CP43; WT, T. elongtus strin with His-tg on the C terminus of CP43 nd in whih the ps 1 nd ps 2 genes re deleted; EPR, Eletron Prmgneti Resonne; DCMU, 3-(3,4-dihlorophenyl)-1, 1-dimethylure; PQ, plstoquinone 9; D2-Y160F, site-direted mutnt without the tyrosine TyrD Corresponding uthors. E-mil ddresses: lin.ouss@e.fr (. Bouss), miw.sugiur@ehime-u..jp (M. Sugiur), frie.rppport@ip.fr (F. Rppport). isolted from the thermophili ynoterium Thermosynehoous elongtus [1 3]. PSII is mde up of 17 memrne protein suunits, 3 extrinsi proteins, 35 hlorophyll moleules, 2 pheophytin moleules, 2 hemes, 1 non-heme iron, 2 (+1) quinones, 4 Mn ions, 1 C 2+ [1 3] nd t lest 1 Cl [3 5], 12 rotenoid moleules nd 25 lipids [3]. sorption of photon y hlorophyll moleule is followed y the trnsfer of the exiton to the photohemil trp nd the onseutive formtion of rdil pir in whih the pheophytin moleule, Pheo D1,is redued nd the hlorophyll moleule, Chl D1, is oxidized [6 8]. The positive hrge is then stilized on P 680, wekly oupled hlorophyll dimer (see e.g. [9 12] for energeti onsidertions). P oxidizes tyrosine residue of the D1 polypeptide, Tyr Z, whih in turn oxidizes the Mn 4 C-luster. The pheophytin nion (Pheo D1 ) trnsfers the eletron to the primry quinone eletron eptor,, whih in turn redues seond quinone, Q B. is tightly ound nd ts s one-eletron rrier wheres Q B ts s two-eletron nd two-proton eptor with stle semiquinone intermedite, Q B.WhiletheQ B semiquinone /$ see front mtter 2010 Elsevier B.V. ll rights reserved. doi: /j.io

2 120. Bouss et l. / Biohimi et Biophysi t 1807 (2011) stte is tightly ound, its quinone nd quinol forms re exhngele with the quinone pool in the thylkoid memrne [13 17]. The Mn 4 C-luster, devie umulting the four oxidizing equivlents required to split wter into dioxygen, is the tive site for wter oxidtion. During the enzyme yle, the oxidizing side of PSII goes through five sequentil redox sttes, denoted S n where n vries from 0 to 4 upon the sorption of 4 photons [18,19]. Upon formtion of the S 4 stte two moleules of wter re rpidly oxidized, O 2 is relesed nd the S 0 -stte is regenerted. The min oftors involved in the funtion of PSII re orne y D1 nd D2 proteins. There re three ps genes enoding the D1 protein in the T. elongtus genome [20]. The omprison of the mino-id sequene dedued from the ps 3 gene to tht dedued from the ps 1 nd ps 2 genes points differene of 21 nd 31 residues, respetively. In T. elongtus, the limtion to high light intensities indues the expression of the ps 3 gene to the detriment of the ps 1 gene [21,22]. We hve proposed tht this regultion t the trnsription level is not mere djustment of the protein synthesis ut rther n limtion t the funtionl level wherey the funtionl properties of PSII re djusted to ope with the inresed photon flux [23]. mongst the 21 mino ids whih differ etween Ps1 nd Ps3 like D1-S270, D1-S153 nd D1-Q130E (see [23] for disussion), the residue t position 130 hs ught muh ttention. It is Gln in Ps1 nd Glu in Ps3. The Gln to Glu exhnge is expeted to stilize the Pheo D1 nion rdil [24 27] nd it hs een shown tht, ordingly, the midpoint potentil of Pheo D1 is inresed y 17 mv from 522 mv in Ps1-PSII [23] to 505 mv in Ps3-PSII [12]. This inrese is out hlf tht found in the single D1-Q130E site-direted mutnt in Synehoystis PCC 6803 [24 27]. This led us to propose tht the effets of the D1-Q130E sustitution ould e, t lest prtly, ompensted for y some of the dditionl mino-id hnges ssoited with the Ps3 for Ps1 sustitution [23]. The eletron trnsfer rte etween P nd Y Z ws lso found fster in Ps3 thn in Ps1 whih suggests hnge in either the midpoint potentil of either Y Z or P 680 /P ouple (nd hene tht of P 680 /P ) or in the reorgniztion energy or less likely in the distne etween P 680 nd Y Z when shifting from Ps1 to Ps3. To ssess the possile differenes of the /Q B inding sites we hve ompred here the inding properties of heriides to the Ps1-PSII nd Ps3-PSII. Two different lsses of ompounds re known to inhiit the reoxidtion of Q y Q B : ure derivtive heriides suh s DCMU nd phenoli heriides suh s romoxynil. These two types of ompounds hve een shown to hve opposite onsequenes on the midpoint potentil of the Q / ouple [28,29].InplntPSII,DCMUinresesthe midpoint potentil of Q / (mking it less negtive), onversely, romoxynil dereses the midpoint potentil of Q / (mking it more negtive) [28,29]. The inding of romoxynil indues derese of the free energy gp etween the 1 [P Q ] nd the 1 [P Pheo D1 ] rdil pirs whih fvors the formtion of the 1 [P Pheo D1 ]yreverse eletron trnsfer nd therefore the formtion of the 3 [P Pheo D1 ]stte nd then 3 P 680.Sine 3 P 680 n ret with oxygen, forming singlet oxygen, speies likely responsile for photodmge, it hs een proposed tht, in plnt PSII, phenoli heriides inrese the sensitivity of PSII to light, while DCMU protets ginst photodmge [28,29]. In T. elongtus, the reltive effets of DCMU nd romoxynil on the midpoint potentil of the Q / ouple in Ps1-PSII were found similr to those in plnt PSII [23]. In ontrst, in Ps3-PSII the thermoluminesene of the S 2 Q hrge reomintion nd the C N virtionl modes of romoxynil deteted in the redued minus oxidized non-heme iron FTIR differene spetr supported either two inding sites or one inding site with two onformtions [23]. In the present work, the effets of DCMU nd romoxynil inding hve een studied y EPR nd time-resolved UV visile sorption spetrosopies in Ps1-PSII nd Ps3-PSII from T. elongtus. Itis shown tht 1) the ffinity of DCMU nd romoxynil for PSII in the stte strongly differs, onsistent with the respetive shifts undergone y the midpoint potentil of / in the presene of these inhiitors, nd 2) the ffinity of DCMU nd romoxynil for PSII in the Fe III Q B stte differs in Ps1-PSII nd Ps3-PSII whih indites different midpoint potentil of the non-heme iron in Ps1-PSII nd Ps3- PSII. Moreover, the oservtion of DCMU-indued oxidtion of the non-heme iron strongly suggests tht Fe III is the Q 2 eletron eptor previously reveled y Joliot nd Joliot [30,31]. 2. Mterils nd methods The iologil mteril used ws His-tgged PSII ore omplexes purified from 1) T. elongtus WT ells, strin in whih oth the ps 1 nd ps 2 genes hve een deleted nd whih therefore only expresses ps 3 [32], 2) 43H ells [33] whih express the ps 1 gene [23,34] nd 3) the D2-Y160F ells [35] whih re expeted to lso express the ps 1 gene. PSII purifition ws done s previously desried [23,32]. sorption hnges were mesured with l-uilt spetrophotometer [36] where the sorption hnges re smpled t disrete times y short flshes. These flshes were provided y neodymium: yttrium luminum grnet (Nd:YG, 355 nm) pumped optil prmetri osilltor, whih produes monohromti flshes (1 nm fullwidth t hlf-mximum) with durtion of 5 ns. Exittion ws provided y dye lser (685 nm, mj) pumped y the seond hrmoni of Nd:YG lser. The pth length of the uvette ws 2.5 mm. PSII ws used t 25 μg of Chl ml 1 in 10% glyerol, 1 M etine, 15 mm CCl 2, 15 mm MgCl 2, nd 40 mm MES (ph 6.5). PSII were drk-dpted for 1 h t room temperture (20 22 C) efore the ddition of either 0.1 mm phenyl-p-enzoquinone (PPBQ, dissolved in ethnol) or 0.1 mm DCMU or 0.1 mm romoxynil (dissolved in ethnol). Cw-EPR spetr were reorded using stndrd ER 4102 (Bruker) X-nd resontor with Bruker Elexsys 500 X-nd spetrometer equipped with n Oxford Instruments ryostt (ESR 900). Flsh illumintion t room temperture ws provided y Nd:YG lser (532 nm, 550 mj, 8 ns Spetr Physis GCR ). PSII smples t 1.1 mg of Chl ml 1 were loded in the drk into qurtz EPR tues nd further drk-dpted for 1 h t room temperture efore eing frozen in the drk to 198 K in dry-ie ethnol th nd then trnsferred to nd stored in liquid N 2. Prior to the mesurements, the smples were trnsferred in the drk t 198 K in dry-ie ethnol th nd degssed s lredy desried [37]. The ddition of PPBQ (0.5 mm, finl onentrtion), DCMU nd romoxynil, dissolved in ethnol 95% ws done in the drk. For the experiments reported in Figs. 1 nd 2, the dditions were done fter the reording of the drk spetr. For the experiments reported in Figs. 3 to 9, the dditions were done fter the drk-dpttion. The DCMU nd romoxynil onentrtions in stok solutions were djusted so tht the finl ethnol onentrtion ws 3% in ll the onditions studied. 3. Results Fig. 1 illustrtes in detil the protool used in this study. The spetr were reorded in Ps3-PSII t 4.2 K in the sene (, lk spetrum) nd presene (, red spetrum) of PPBQ (Fig. 1), of DCMU (Fig. 1B) nd of romoxynil (Fig. 1C). Spetr (in lue) re the differene spetr spetrum -minus-spetrum in eh orresponding pnel. In ddition to the spetrosopi fetures sensitive to the ddition of quinone nlogues or heriides whih will e desried elow, the spetrum of the drk-dpted smples shows the following fetures: i) the g z nd g y resonnes of Cyt 550 nd to less extent of Cyt 559 t 2230 G nd 2920 G, respetively [38 40], whih re strongly sturted owing to the temperture nd the mirowve power onditions used here, ii) signl t g=4.3 ( 1600 G) nd t g vlues 5 (etween 500 nd 1500 G) from ontminnts Fe III (whih should

3 . Bouss et l. / Biohimi et Biophysi t 1807 (2011) g vlue g vlue B C Fig. 1. EPR spetr reorded in Ps3-PSII t 4.2 K. Effet of the ddition of PPBQ (pnel ), of DCMU (pnel B), nd of romoxynil (pnel C). Spetr (lk spetr) were reorded in drk-dpted PSII, spetr (red spetr) were reorded fter the ddition of either PPBQ or DCMU or romoxynil nd spetr (lue spetr) re the differene spetr fterminus-efore the ddition. Other instrument settings: [Chl]=1.1 mg ml 1 ; modultion mplitude, 25 G; mirowve power, 20 mw; mirowve frequeny, 9.4 GHz; nd modultion frequeny, 100 khz. The Tyr D spetrl region t g 2 ws deleted. Fig. 2. EPR spetr reorded in D2-Y160F PSII t 4.2 K. The spetrum shown is the differene spetrum efore-minus-fter the ddition of PPBQ. The sme other instrument settings s in Fig. 1. not e mistken with the non-heme iron signl studied elow), nd iii) n out-off-sle nrrow signl t g 2.0 ( 3400 G) from the Tyr D whih is deleted in the spetr shown. None of these signls were ffeted y the ddition of the PPBQ or the heriides. The ddition of PPBQ (Fig. 1) resulted in the dispperne of the signl ssigned to Fe II Q B, s shown y the negtive Fe II Q B signl in spetrum etween 3200 G nd 4500 G, inditing tht the drkdpted Ps3-PSII ontined signifint mount of Q B. The ddition of PPBQ lso indued the pperne of the non-heme Fe III signl minly deteted y the two hrteristi spetrl fetures t g =5.65 ( 1200 G) nd g=7.5 ( 900 G). s disussed in [41 43], the oxidnt of the non-heme Fe II is the semiquinone PPBQ formed upon the redution of PPBQ y Q B :Fe II +PPBQ +2H + Fe III +PPBQH 2. The ddition of DCMU (Fig. 1B) lso resulted in the dispperne of the signl ssigned to Q B. In ddition, it indued the pperne of severl fetures (t G, see spetrum ) whih reflet the formtion of t the expense of Q B in greement with the finding tht DCMU shifts to the right hnd term the following equiliri: Fe II Q B +DCMU Fe II Q B +DCMU Fe II DCMU+Q B [17,44], see lso se(2) in Chrt 1. The differene spetrum in the spetrl rnge of the non-heme Fe III inludes derivtive shped signl whih reflets the well-known DCMU-indued hnges of the zero-field splitting prmeter of the S=5/2 stte of the non-heme Fe III [43]. ssuh,this signl shows tht the non-heme iron is oxidized in low proportion of drk-dpted Ps3-PSII even in the sene of ny ddition. lthough one would expet tht the ddition of romoxynil (Fig. 1C) indues s well modifition of non-heme Fe III signl, these hnges were smller thn those oserved upon the ddition of DCMU (ompre spetr in Fig. 1C nd B) nd this will e ddressed elow. The spetrl hnges deteted in the quinone region etween 3200 G nd 4500 G upon the ddition of romoxynil re of smller mplitude thn the DCMU-indued ones. Moreover, we oserved tht the ddition of ethnol lone resulted in similr spetrl hnges (not shown) so tht we ssign the spetrl hnges in Fig. 1C in the G region to solvent effet rther thn to displement of the Fe II Q B + romoxynil Fe II Q B +romoxynil Fe II romoxynil+q B equiliri. Consistent with this, romoxynil ddition indued no signifint spetrosopi hnges ttriutle to the formtion of suggesting tht romoxynil does not ind to Ps3-PSII in whih the Q B stte is present. The out-of-sle signl from the TyrD rdil overlps with the signl rising from the Fe II Q B nd thus preludes its full hrteriztion. We

4 122. Bouss et l. / Biohimi et Biophysi t 1807 (2011) g vlue g vlue B C Fig. 3. EPR spetr reorded in Ps3-PSII t 4.2 K. Spetr were reorded in the presene of 3% ethnol (pnel ), in the presene of DCMU (pnel B), nd in the presene of romoxynil (pnel C). Spetr were reorded in drk-dpted smples (, lk spetr) nd fter one flsh given t room temperture (, red spetr). Spetr (lue spetr) re the differene spetr fter-minus-efore the ddition. The sme other instrument settings s in Fig. 1. The Tyr D spetrl region t g 2 ws deleted. Fig. 4. EPR spetr reorded in Ps3-PSII t 8.6 K. Spetr were first reorded in drkdpted smples nd then fter one flsh given t room temperture. The spetr shown re the light-minus-drk spetr in the presene of 3% ethnol (), of DCMU (), nd of romoxynil (). The sme other instrument settings s in Fig. 1. The Tyr D spetrl region t g 2 ws deleted. thus repeted the sme experiment (Fig. 2)stheoneshowninFig. 1 with the D2-Y160F mutnt [35]. In these onditions, the differene spetrum efore-minus-fter the ddition of PPBQ (spetrum )exhiits the omplete Fe II Q B EPR signl whih is detetle from 3000 to 5000 G. This signl will e desried with more detils y Sedoud et l. (mnusript sumitted). t this stge two mjor onlusions n e drwn: i) signifint frtion of Ps3-PSII is in the Q B stte in the drk, ii) wheres DCMU inding indues the formtion of t the expense of Q B, romoxynil does not t lest up to onentrtion of 200 μm. These onlusions my e further vlidted y mesuring the flsh-indued hnges in the redox stte of the vrious eletron rriers in the PSII eptor side, s illustrted in Fig. 3 whih shows the flsh-indued EPR spetr in the presene of ethnol (the solvent used here) (Fig. 3) or DCMU (Fig. 3B) or romoxynil (Fig. 3C). fter ddition of these hemils, the spetr were reorded t 4.2 K prior to (spetr ) nd fter (spetr ) the illumintion y 1 flsh t room temperture. Spetr re the differene spetr fter-minus-efore the flsh illumintion. lthough the flsh illumintion should lso indue the formtion of the S 2 -stte, this one is strongly sturted, owing to the temperture nd the mirowve power onditions used here, whih prevents n urte estimtion of its mplitude in these onditions. In the presene of ethnol (Fig. 3), we hrdly deteted ny spetrl hnges etween 3200 nd 4500 G upon the flsh illumintion other thn the strongly sturted S 2 multiline signl. This suggests tht the proportion of the enters in the Fe II Q B stte ws not modified y the flsh illumintion, or tht, in other words, there ws pproximtely 50% of Ps3-PSII with Fe II Q B in the drk-dpted stte. The lk of Fe II formtion in the sene of ny rtifiil eletron eptor is in greement with previous estimtes in whih t lest 2 or 3 Q B moleules re present per retion enter in PSII from T. elongtus [3,45 47]. We noted ove tht frtion of PSII enters re in the non-heme Fe III stte, even in the sene of ny ddition. Expetedly, the light-indued hrge seprtion leds to the redution of the non-heme Fe III s witnessed y the negtive non-heme Fe III signl with negtive fetures t g=5.65 ( 1200 G) nd g=7.5 ( 900 G) in spetrum in Fig. 3. The results in Fig. 3B show tht fter the ddition of DCMU the flsh illumintion indued the formtion of signl with feture t g 1.9 ( 3500 G) whih is hrteristi of Fe II. The semiquinone rdil is mgnetilly oupled to the high spin (S=2) non-heme

5 . Bouss et l. / Biohimi et Biophysi t 1807 (2011) g vlue ΔΙ/Ι x B Time (ms) Fig. 6. Kinetis of the sorption hnges t 320 nm in Ps3-PSII. Blk irles were reorded fter the first sturting flsh. For the red irles, mesurements were done 500 μs nd 6 ms fter the first flsh then seond sturting flsh ws given 100 ms fter the first one. The ontinuous lue line is the dey fter the seond flsh normlized to the sme time sle nd the sme mplitude s tht of the first flsh. The smples (Chl=25 μg ml 1 ) were drk-dpted for 1 h t room temperture efore the ddition of 100 μm DCMU Fig. 5. EPR spetr reorded in Ps3-PSII t 4.2 K (pnel ) nd t 8.6 K (pnel B). Spetr were reorded in the presene of 3% ethnol (spetr ), in the presene of DCMU (spetr ), nd in the presene of romoxynil (spetr ). Spetr were reorded in drk-dpted smples nd fter 198 K illumintion. The spetr shown re the light-indued spetr. The sme other instrument settings s in Fig. 1. TheTyr D spetrl region t g 2 wsdeleted. Fe II Q B +heriide Fe III Q B H 2 +heriide Fe III heriide+ Q B H 2 to the right, suggesting tht romoxynil hs lower ffinity thn DCMU for the enters in the Fe III stte. ll the results ove nd elow re summrized in Chrt 2. ll the spetr shown ove were reorded t 4.2 K, temperture whih llows the detetion of the non-heme iron signl nd tht of the quinone-fe II signls with lmost no overlpping from the S 2 multiline signl. Yet, the S 2 multiline signl, eing extremely speifi of the dvnement of the S-stte yle, is relile wy to quntify the frtion of PSII enters in whih hrge seprtion hs ourred. By doing so, one n quntify the frtion of PSII enters in the stte prior to the flsh nd ssess this quntity in the presene of either 1.0 ferrous ion whih is loted etween nd Q B [1 3] whih gives rise to the rod EPR signls with turning points round g 1.8 nd 1.9, e.g. [45] nd referenes therein. In priniple, this signl n serve s mrker of the frtion of PSII enters with n oxidized Q B efore the ddition of DCMU, sine DCMU inding indues the formtion of Q Fe II in the drk in those enters in the Q B stte efore ddition. Yet, the piture my e slightly more omplited thn tht. Indeed, spetrum in Fig. 3B shows tht the negtive signl in the non-heme Fe III region ws lrger in the presene of DCMU thn in its sene. This indites tht the frtion of enters in the non-heme Fe III stte in the drk is lrger when DCMU is ound or tht, in other terms, in ddition to the Fe II Q B +DCMU Q Fe II Q B +DCMU Q Fe II DCMU+ Q B equiliri, the following equiliri Fe II Q B +DCMU Fe III Q B H 2 + DCMU Fe III DCMU+Q B H 2 might our in Ps3-PSII. The results in Fig. 3C show tht, in the presene of romoxynil, the flsh-indued signl ssigned to Q Fe II ws smller thn in the presene of DCMU. Moreover, in ontrst to the DCMU se, the negtive signl in the non-heme Fe III region ws similr in the presene of romoxynil thn in its sene. This indites tht romoxynil inding does not ffet the redox stte of the non-heme iron. Thus, t vrine with DCMU, romoxynil does not shift the equiliri (ΔI/I 1 (t)-δ I/I 2 (t))/δ I/I 2 (100ms) Time (µs) Fig. 7. Kinetis of the sorption hnges t 552 nm in Ps3-PSII fter the first sturting flsh of sequene. The Y-xis is the differene etween the mplitude of the ΔI/I mesured t the indited time fter the first nd seond flses divided y the mplitude of the ΔI/I mesured 100 ms fter the seond flsh. The smples (Chl=25 μg ml 1 ) were drk-dpted for 1 h t room temperture efore the ddition of 100 μm DCMU. The fit ws done with n exponentil dey with n offset with OriginPro softwre (OriginL Corportion).

6 124. Bouss et l. / Biohimi et Biophysi t 1807 (2011) [PSII-Bromoxynil], µm [Bromoxynil], µm Fig. 8. [PSII-romoxynil] versus the totl romoxynil onentrtion (lk symols). The [PSII-romoxynil] onentrtion ws estimted y mesuring the sorption hnges t 552 nm fter sturting flsh t 10 μs nd 10 ms nd then y pplying the following reltion: [PSII-romoxynil]=(ΔI/I 10 μs +romox ΔI/I 10 ms +romox)/(δi/i 10 μs +romox). The smples (Chl=25 μgml 1 =0.8 μm) were drk-dpted for 1 h t room temperture efore the ddition of romoxynil t the indited onentrtions. The fitting proedure ws done with OriginPro softwre ssuming single inding site with the dissoition onstnt K d =[PS II] free [I] free /[PSII-I] with I for romoxynil. fter the strightforwrd sustitutions we hve: Kd*[PSII-I]=([PSII] totl *[I] totl ) ([PSII] totl *[PSII-I]) ([I] totl * [PSII-I])+[PSII-I] 2. DCMU or romoxynil. We thus reorded the spetr t 8.6 K, with mirowve power of 20 mw, to detet the S 2 multiline signl in T. elongtus PSII. This signl is entered t g=2 with t lest 18 lines sped y 90 G from 2500 to 4500 G nd rises from n eletroni spin S=1/2 ground stte in mgneti intertion with the nuler spin I=5/2 of the 4 Mn ions [48 50]. Fig. 4 shows the light-minus-drk spetr reorded in Ps3-PSII t 8.6 K fter flsh illumintion in the presene of ethnol (spetrum ), in the presene of DCMU (spetrum ) or romoxynil (spetrum ). s for the spetr mesured t 4.2 K, the spetr mesured t 8.6 K show tht the non-heme Fe III signl whih disppered upon the flsh illumintion g vlue Fig. 9. EPR spetr reorded in Ps1-PSII t 8.6 K. Spetr were first reorded in drkdpted smples nd then fter one flsh given t room temperture. The spetr shown re the light-minus-drk spetr in the presene of 3% ethnol (), of DCMU (), nd of romoxynil (). The sme other instrument settings s in Fig. 1. The Tyr D spetrl region t g 2 ws deleted [PSII-Bromoxynil]/[PSII totl], % hd similr mplitude in the presene of romoxynil nd ethnol ut lrger one in the presene of DCMU. The flsh-indued S 2 -multiline signl in the presene of DCMU nd romoxynil ws pproximtely hlf tht in the presene of ethnol lone. This is strightforwrdly explined in the DCMU se sine 50% of the Ps3-PSII enters re in the losed Q Fe II DCMUstteeforetheflsh illumintion. In the romoxynil se, we hve shown ove tht the fte of the enters in the Fe II Q B se (whih we estimted to e 50%) differs. Indeed, Fig. 1C showedtht romoxynil ddition does not result in the formtion of the Q Fe II stte t the expense of Fe II Q B nd tht the reltive mount of the ltter stys unhnged. Thus one would expet to oserve the formtion of the S 2 -stte in the mjority of the enters, if not ll. However, we lso noted erlier, tht the reltive mount of Q Fe II formed y the flsh illumintion (Fig. 3) is smller in the presene of romoxynil thn in the presene of DCMU, suggesting tht, under the onditions of the experiment, hrge reomintion ours nd leds to derese of the detetle mount of S 2 Q stte. In reent work, we hve shown tht the hrge reomintion etween S 2 nd Q Fe II in the presene of romoxynil t room temperture ours in the seond time rnge in Ps3-PSII [23]. The drk time etween the flsh illumintion nd the freezing of the smple ( 1 2s)isthuslongenoughtollowthe ourrene of the S 2 Q Fe II hrge reomintion in signifint proportion of the enters. The dt shown elow shows tht suh is indeed the se. Fig. 5 shows the light-minus-drk spetr reorded in Ps3-PSII t 4.2 K (pnel ) nd t 8.6 K (pnel B) fter 198 K illumintion in the presene of either ethnol (spetr ), or DCMU (spetr ) or romoxynil (spetr ). For reson of lrity, the spetrl rnge shown from 2500 to 5000 G is foused on tht inluding the S 2 -multiline signl nd the semiquinone signls. The spetr reorded t 4.2 K (pnel ) will e desried in first. In the presene of ethnol only (spetrum ), the light-indued signl exhiited lrge feture entered t g 1.6 ( 4200 G) whih is hrteristi of the Q Fe II Q B stte e.g. [45,51]. This signl ws formed in the enters with the Fe II Q B stte present efore the 198 K illumintion sine the eletron trnsfer from to Q B is loked t suh temperture. The Q Fe II Q B stte, whih is expeted to e formed in the other enters, is lso deteted t g 1.9 ( G) e.g. [45,51,52]. Nevertheless the smll mplitude of the Q Fe II signl in these EPR onditions together with the overlpping of the Q Fe II Q B signl whih is intrinsilly muh more intense mkes the detetion of the Q Fe II signl diffiult. In the presene of DCMU (spetrum ) only the formtion Q Fe II signl t g 1.9 ws deteted (with the min feture etween 3500 nd 3700 G), showing tht there were no remining enters in the Fe II Q B prior to the 198 K illumintion. In the presene of romoxynil (spetrum ) the light-indued g=1.6 signl from Q Fe II Q B hd similr mplitude to tht reorded in the sene of the heriide (spetrum ). This gin shows tht in the enters with the Fe II Q B stte efore the ddition of romoxynil, the ddition of this heriide did not result in the formtion of the Q Fe II romoxynil stte. The Q Fe II signl in spetrum rises from enters in the Fe II Q B stte prior to the 198 K illumintion. Pnel B in Fig. 5 shows the differene spetr indued y the 198 K illumintion nd reorded t 8.6 K. In the sene of heriide (spetrum ) the S 2 -multiline signl is now fully deteted in ddition to the Q Fe II Q B signl. In the presene of DCMU (spetrum ) the mplitude of the S 2 -multiline signl ws pproximtely hlf of tht in spetrum onsistent with the estimte ove tht 50% of the enters were in the Q Fe II DCMU stte efore the 198 K illumintion. In the presene of romoxynil (spetrum ), the mplitudes of oth the Q Fe II Q B signl nd the S 2 -multiline signl were similr to those oserved in the sene of the heriide, showing tht the smller mplitude of the S 2 -multiline signl in the experiment fter illumintion y one flsh nd shown in Fig. 3 ws due to fst S 2 hrge reomintion whih is prevented in the 198 K illumintion proedure.

7 . Bouss et l. / Biohimi et Biophysi t 1807 (2011) K Q K i ox Q B + I + I I (1) K et K i red Q Q B + I Q B + I I K Q (2) + I K I ox I K K i (3) K I red I I Chrt 1. Thermodynmi equiliri desriing the ses 1, 2 nd 3 disussed in the text. We hve shown ove tht the non-heme iron, whih is potentil eletron eptor in the Fe III stte, is oxidized in smll proportion of Ps3-PSII nd tht this proportion inreses in the presene of DCMU. This effet of DCMU ws further investigted in the experiment reported in Fig. 6. In Fig. 6, the sorption hnge in Ps3-PSII ws followed fter the first nd seond sturting flshes given fter the ddition of DCMU to the drk-dpted smple. The mesurement ws done t 320 nm wvelength whih orresponds to mximum in the Q -minus- differene spetrum nd t whih the S 2 -minus-s 1 differene spetrum lso ontriutes e.g. [53 57]. Thefilled lk irles show the dey of the light-indued sorption hnge fter the first flsh. The first point +DCMU (50-x)% S 1 Fe II /DCMU ( x+y)% S 1 Fe III /DCMU (50-y)% S 1 Fe II /DCMU 1f (50-x)% S 2 Q Fe II /DCMU (x+y)% S 2 Fe II /DCMU (50-y)% S 1 Q Fe II /DCMU 50% S 1 Fe II Q B (50-x)% S 1 Fe II Q B x% S 1 Fe III Q B +Bromoxynil 50% S 1 Fe II Q B (50-x)% S 1 Fe II /Bromox x% S 1 Fe III Q B 1f 50% S 2 Fe II Q B (50-x)% S 2 Fe II /Bromox x% S 2 Fe II Q B (50+x)% S 2 Fe II /Bromox (50-x)% S 2 Fe II /Bromox Chrt 2. Shemti events following the first flsh given on drk-dpted PSII in the presene of either DCMU or romoxynil. Centers in the S 0 stte re not onsidered ut they likely ehve like those in the S 1 stte. ws mesured 500 μs fter the flsh. The red filled irles show the sorption hnges mesured 500 μs nd6 msfterfirst flsh nd then the dey ws followed fter the seond flsh. The ontinuous lue line is the dey reorded fter the seond flsh (red points) ut with time sle nd mplitude sle resled to tht reorded fter the first flsh. The kinetis of the dey fter the seond flsh ws similr to tht fter the first flsh. The S 2 Q hrge reomintion in the presene of either DCMU or romoxynil ws reently studied in detil in oth Ps3-PSII nd Ps1-PSII [23]. Therefore, for the present purpose we will fous minly on the mplitudes nd less on the kinetis. The min differene etween the lk nd red tres is the mplitude of the signl whih is lrger fter the seond flsh thn fter the first flsh. In the ontext of the model proposed ove (see Chrt 2) the results in Fig. 6 suggest tht is formed in ll the enters fter the first flsh nd reoxidized in less thn 500 μs y the non-heme iron in the frtion of the enters in the Fe III stte prior to the flsh illumintion. In plnt PSII, the hlf-time of this kinetis ws mesured to e 25 μs [42] ut is unknown in T. elongtus smples. Therefore, this kinetis ws mesured in our smple nd the result is shown in Fig. 7. In Fig. 7,Q formtion nd reoxidtion ws followed t 552 nm in Ps3-PSII s in [42]. This wvelength orresponds to the trough of the pheophytin nd shift oserved upon redution [58 60] nd is lmost free from other spetrl ontriutions, whih is not the se t 320 nm [53 57]. The mplitude mesured 10 μs fter the flsh of the signl ws similr to tht mesured fter the seond one (not shown). This shows tht Q is indeed formed in ll the enters fter one sturting flsh. Nevertheless, wheres the signl did not dey in the hundred μs time sle fter the seond flsh, the signl deyed with hlf-time lose to 55 μsin 20% of the enters fter the first flsh, nd we ssign this dey to the Q Fe III DCMU Fe II DCMU retion. It is shown ove tht the inding of romoxynil only ourred in enters in the Q B stte. To estimte the dissoition onstnt of romoxynil for the Q B poket in Ps3-PSII we hve rried out the following experiment. The sorption hnges were mesured t 552 nm (s in Fig. 7, [42])10μsnd 10 ms fter one flsh. The mplitude t 10 μs orresponded to the mximum mount of Q indued y one flsh nd ws similr for ll the romoxynil onentrtions used (not shown). From 10 μs to 10 ms the signl deyed in enters in whih romoxynil ws not ound. This dey is due to the eletron trnsfer from Q to Q B. The proportion of enters in whih romoxynil ws ound ws therefore estimted y the following reltion: [PSIIromoxynil]=(ΔI/I 10 μs +romox ΔI/I 10 ms +romox)/(δi/i 10 μs +romox). Fig. 8, filled irles, shows the [PSII-romoxynil] onentrtions s estimted ove versus the totl romoxynil onentrtion together with fit (ontinuous line) ssuming one inding site. The PSII onentrtion ws 25 μg Chlml 1 whih is equivlent to 0.8 μm. The

8 126. Bouss et l. / Biohimi et Biophysi t 1807 (2011) fitting proedure resulted in dissoition onstnt equl to 2.3 μm nd inding of romoxynil in 70% of the enters whih suggests tht in this experiment Q B ws present in 30% of the enters. ll the results shown ove were otined in Ps3-PSII. The results elow now del with Ps1-PSII. In the experiment reported in Fig. 9, the Ps1-PSII smples were illuminted y one flsh t room temperture in the presene of either ethnol (spetrum ) or DCMU (spetrum ) or romoxynil (spetrum ). Spetr were reorded t 8.6 K. It is noteworthy tht the S 2 Q romoxynil hrge reomintion is slower in Ps1-PSII thn in Ps3-PSII [23] so tht, s shown elow, it ws unneessry to derese the temperture t whih the smples were illuminted to void hrge reomintion. The mplitudes of the S 2 -multiline signl indued y one flsh given t room temperture with nd without romoxynil were similr wheres the S 2 -multiline signl ws pproximtely hlf in the presene of DCMU. This shows tht s for Ps3-PSII, romoxynil is unle to ind in enters in the Q Fe II stte in Ps1-PSII. This is further supported y the similr mplitude of the Fe II Q B EPR signl in Ps1-PSII efore nd fter the ddition of romoxynil (not shown). Comprison of the mplitude of the S 2 -multiline signl in the presene of DCMU with tht in the two other onditions indites tht Fe II Q B ws lso present in 50% of drk-dpted Ps1-PSII. Fig. 9 lso shows tht the mplitude of the non-heme Fe III EPR signl whih disppers upon flsh illumintion is similr in the 3 smples. This indites tht 1) s in Ps3-PSII, there is proportion of enters with n oxidized non-heme iron in the drk-dpted stte in the sene of ny ddition ut 2) in ontrst to Ps3-PSII, the ddition of DCMU to Ps1-PSII did not indue further inrese in the proportion of non-heme Fe III. ll the results otined in the Ps1-PSII were lso otined in the D2-Y160F smple (not shown) whih is expeted to express the ps1 gene under our growth onditions sine it ws onstruted in strin with the 3 ps genes. 4. Disussion The inding of ompetitive inhiitor to the plstoquinone Q B poket requires the ltter to e, t lest trnsiently, empty [17,44]. On the sis of these premises, Lvergne nd Velthuys distinguished two different ses depending on the redox stte of Q B in the drk. When Q B is oxidized (se (1) in Chrt 1), the respetive inding proilities of plstoquinone nd of the inhiitor will e determined y the ompetition etween these two, i.e. y their respetive ffinity for the site nd their onentrtion. The presene of Q B in its semiquinone stte (se (2) in Chrt 1) does not prevent the inding of the inhiitor sine the neutrl stte Q B n e formed vi the equilirium etween Q B nd Q Q B, yet it is expeted to derese the pprent ffinity of the inhiitor for its site owing to the uphill hrter of this equilirium. The ddition of n inhiitor of the Q B poket is expeted to disple the equilirium etween Q B nd Q Q B in fvor of the ltter nd hene to indue the formtion of Q. These different expettions re stisfyingly met in the se of DCMU in plnt PSII [44]. s disussed in [17,44] nd doumented in the Supplementry mterils, the pprent dissoition onstnt of the inhiitor, whih tkes into ount the ompetition of the oupny of the Q B poket etween plstoquinone nd the inhiitor, is expeted to depend on the redox stte of. Importntly, this sttement holds even when the intrinsi dissoition onstnts of the inhiitor, defined ording to ses (1) nd (2) s K ox i =[ ][I]/[ I] nd K red i =[Q ][I]/[Q I], [I] eing the free heriide onentrtion, re equl, or in other terms when the inding onstnt of the inhiitor to the empty Q B site does not depend on the redox stte of one otins: K ox pp =K red pp /(1+K et )(see Supplementry mteril). K et hs een estimted to e 20 [61], so tht the dissoition onstnt/ffinity onstnt of the inhiitor is expeted to e out 20 fold lrger/smller when is in its semiquinone stte. Wheres this is indeed the se for DCMU here, s in plnt PSII [44],wefoundtht,inthe se of romoxynil, the derese in the pprent ffinity onstnt for the Q stte is muh lrger thn the ove expettion. Indeed, the present results lerly indite tht in those enters in whih Q B is present prior to the ddition of the heriide, the ddition of DCMU results into the dispperne of the Fe II Q B EPR signl nd into the formtion of the EPR signl hrteristi of Q Fe II. In ontrst, upon the ddition of romoxynil, the Fe II Q B EPR signl is not ffeted nd no EPR signl from Q Fe II is deteted. This indites tht n dditionl prmeter prtiiptes to the modultion of the inding onstnt of romoxynil y the redox stte of. s we will now disuss, the romoxynil-indued shift of the midpoint potentil of the Q / ouple is likely ndidte. If one now neglets, temporrily nd for the ske of simpliity, the ompetition etween the inhiitor nd the quinone inding nd only onsider the reltionship etween the redox stte of nd the inding properties of the inhiitor the Q B poket, one n desrie the relevnt equiliri s desried in se (3) of Chrt 1. ording to suh squre digrm, the two midpoint potentils of the Q i / ouple in the presene (E m ) nd in the sene (E m )of the heriide re linked y the following reltionship [62,63]: E i m = E m + RT nf ln! Kred i Ki ox : This reltion shows tht different ffinities of the heriide for the or Q sttes respetively should trnslte into different midpoint potentils in the presene nd sene of heriides nd vie vers. In ddition, it shows tht lrger (smller) ffinity for the semiquinone stte thn tht for the oxidized stte trnsltes into n up-shift (downshift) of the midpoint potentil upon heriide inding nd vie vers. Krieger nd ollegues hve shown thn DCMU nd romoxynil hve opposite effet in this respet [28,29]. Wheres DCMU inding results in n up-shifted midpoint potentil of the Q / ouple, romoxynil inding indues down-shift. ssuming t first tht the mplitude of the down-shift is similr in T. elongtus PSII thn in spinh PSII, the 45 mv down-shift would e equivlent to 6 fold lower ffinity of romoxynil for the Q B site in the Q stte thn in the stte. This, omined with the ompetition etween quinone nd romoxynil inding, is expeted to yield 6* 20=120 fold smller ffinity for the semiquinone stte. We found here tht the dissoition onstnt for romoxynil is 2.3 μm when is oxidized. Thus, ssuming similr shift in the midpoint potentil s the one oserved in spinh PSII, the pprent dissoition onstnt when is in the semiquinone stte would e 280 μm. We note tht with 200 μm romoxynil we oserved no detetle inding in the Q stte, so tht the down-shift of the midpoint potentil of the Q / ouple upon the inding of romoxynil is proly lrger thn 45 mv in T. elongtus PSII. It should e noted tht lrger onentrtion rnge ould not relily e investigted owing to soluility prolems nd to seondry effets of romoxynil on the Mn 4 luster (minly the pperne of some Mn 2+, not shown). Importntly, the ove resoning should lso pply to DCMU inding. In other words, the reported up-shift of the midpoint potentil of the Q / ouple oserved upon DCMU ddition is expeted to trnslte into lrger ffinity for DCMU when is in its semiquinone stte. t odds with this expettion, the presene of Q B hs een found to derese the pprent inding onstnt of DCMU nd, s disussed ove, this deresed pprent inding onstnt hs een rtionlized y the ompetition etween the inding of quinone nd DCMU. n interesting possiility to ount for the pprently ontrditory findings tht (i) DCMU inding up-shifts the midpoint potentil of the Q / ouple (nd thus stilizes the semiquinone stte) nd (ii) DCMU inding to PSII in the Q stte is weker, is tht the inding onstnt of quinone to the Q B poket lso depends on the redox stte of. If indeed the inding onstnt of quinone lso inreses signifintly in the presene of the Q, it my ompenste the inresed ffinity for DCMU.

9 . Bouss et l. / Biohimi et Biophysi t 1807 (2011) The ove disussed finding tht the inding onstnt of romoxynil is strongly deresed in the presene of Q rises the issue of the effiieny of this ompound s n inhiitor of the Q B poket. Indeed, PSII enters in the Q B stte in the drk ind romoxynil ording to se (1) in Chrt 1 with rther low dissoition onstnt ( 2.3 μm, see ove), ut the flsh-indued formtion of the semiquinone Q stte my e expeted to trigger the relese of romoxynil nd hene open the gte for eletron trnsfer. To our knowledge the inding nd relese rtes of romoxynil hve not een determined. It is ler however tht the relese of romoxynil is muh slower thn the hrge reomintion etween S 2 nd Q sine otherwise Q would dey vi forwrd eletron trnsfer to Q B rther thn kwrd eletron trnsfer to S 2. The ft tht suh is not the se is supported y severl findings suh s the down-shifted thermoluminesene glow urve oserved in the presene of romoxynil with respet to the DCMU se [23] or the onomitnt dey of S 2 nd Q found when ompring kineti dt monitoring either one of the two prtners, this work nd [23]. The S 2 Q hrge reomintion rte in the presene of romoxynil eing 0.8 s 1 t 25 C in the Ps3 PSII [23], if we ssume the dissoition onstnt to e lrger thn 250 μm (see ove) the inding rte of romoxynil (t 100 μm) would e smller thn 0.3 s 1 in the presene of Q. This figure is smller thn the one found for DCMU ( 5s 1 t the sme onentrtion [44]). The present work is in greement with previous report whih mentioned tht the exhnge of phenoli-type nd ure-type heriides in the Q B site of spinh thylkoids ourred in the seond time rnge [64] nd tht the exhnge ws fster for phenoli heriides thn for ure-type heriides. It hs lso een found tht in spinh thylkoids the dissoition onstnt of romoxynil ws lrger in reduing onditions (K d =220 nm) thn in oxidizing onditions (K d =45 nm) [65], despite ftor 10 in K d vlues etween these vlues nd those reported here, whih re diffiult to rtionlize differently thn y invoking speies dependene. In ddition to the ove onlusion tht the inding onstnt of romoxynil nd DCMU is ffeted y the redox stte of, the present results show tht the ddition of DCMU to enters in the Fe II Q B stte indues the formtion of the Fe III Q B stte in frtion of enters, this frtion eing lrger in Ps3-PSII thn in Ps1-PSII (Fig. 3). This oservtion n lso e explined in the frmework of squre thermodynmi digrm suh s the one depited in se (3) in Chrt 1 with the Fe II /Fe III redox ouple in ple of the Q / ouple. In this model, the midpoint potentils, E m, of the Fe II /Fe III ouple in the presene nd in the sene of the heriide re lso expeted to differ if indeed the inding onstnt of DCMU depends on the redox stte of the non-heme iron. In support to this proposl, Wright found tht the redox stte of the non-heme iron modultes the inding of DCMU [66]. The results in Figs. 3 nd 4 lso show tht upon the ddition of DCMU to Ps3-PSII smple, the heriide-indued Fe III EPR signl orresponds to stte in whih the heriide is ound wheres no extr Fe III EPR signl ws indued y the ddition of romoxynil. Therefore the sme resoning s ove leds us to suggest tht for the Fe II /Fe III + ouple: E DCMU + m E romox. m in Ps3-PSII. In ontrst to Ps3-PSII, the ddition of either DCMU or romoxynil to Ps1-PSII did not result in n extr-oxidtion of the non-heme iron. This suggests tht the midpoint potentil of the nonheme iron differs in Ps3-PSII nd Ps1-PSII eing proly more positive in Ps1-PSII thn in Ps3-PSII t lest when DCMU is ound. This is n dditionl differene etween Ps1-PSII nd Ps3-PSII with respet to those lredy pointed out [23]. From struturl stndpoint, is onneted to the Q B inding poket vi D2-His214-Fe-D1-His215 network through whih the possile struturl hnges resulting from the ouption of the site n propgte to [1 3]. reent FTIR study hs shown tht the frequeny of the CO group of Q is down-shifted in the presene of romoxynil when ompred to DCMU [67]. It is of note however tht the IR nds orresponding to the C=O strething modes of the neutrl hve, until now, remined elusive preluding the hrteriztion of the likely differentil effet of the romoxynil, plstoquinone or DCMU on these modes. In ny se, in the Q stte, the phenolte group of the phenoli heriide forms reltively strong H-ond with the NH group of D1-His215. Importntly, omprtive doking lultions of romoxynil, plstoquinone or DCMU suggest tht the H-ond to this NH groups is stronger with romoxynil thn with PQ nd with PQ thn with DCMU [67]. ordingly, these different oupnts of the Q B poket re expeted to differentilly modify the D2-His214 Fe D1-His215 network nd hene the midpoint potentil of the Q / ouple. Moreover, the non-heme iron eing prt of this network it is lso likely to undergo shift of its midpoint potentil, s proposed here, in response to the wekening of the strength of the H-ond in whih the NH group of D1-His215 is involved, ourring when DCMU reples plstoquinone. The lst point we would like to disuss is the oservtion tht, y induing the oxidtion of the non-heme iron, ddition of DCMU in the drk inreses the numer of potentil eletron eptors on the first flsh. This is reminisent of the results whih led Joliot nd Joliot [30,31] to propose the existene of seond eletron eptor Q 2 in ddition to Q 1,Q 1 eing the other nme of. The eptor Q 2 hs een shown to hve no quinone properties. In the Q 1 /Q 2 model, the existene of n oxidized Q 2 prior to the illumintion prevented the full redution of Q 1. From the results shown in this work, it seems likely tht Fe III, the onentrtion of whih inresing upon the ddition of DCMU in Ps3-DCMU, orresponds to Q 2. In plnt PSII, the hlf-time of the Q Fe III DCMU Fe II DCMU retion ws estimted to e 25 μs [42]. It ws estimted here to e 55 μs in Ps3-PSII in T. elongtus. knowledgements This study ws supported y the JSPS nd CNRS under the Jpn- Frne Reserh Coopertive Progrm; Grnt-in-id for Sientifi Reserh from the Ministry of Edution, Siene, Sports, Culture nd Tehnology ( for M.S.); nd in prt y the EU/Energy Network projet SOLR-H2 (FP7 ontrt ). We would like to thnk Jérôme Lvergne, rezki Sedoud nd Bill Rutherford for helpful disussions nd for ommuniting results prior to pulition. Thnh-ln Li is knowledged for tehnil ssistne. ppendix. Supplementry dt Supplementry dt to this rtile n e found online t doi: /j.io Referenes [1] K.N. Ferreir, T.M. Iverson, K. Mghloui, J. Brer, S. Iwt, rhiteture of the photosyntheti oxygen-evolving enter, Siene 303 (2004) [2] B. Loll, J. Kern, W. Senger,. Zouni, J. Biesidk, Towrds omplete oftor rrngement in the 3.0 Å resolution struture of photosystem II, Nture 438 (2005) [3]. Guskov, J. Kern,. Gdulkhkov, M. Broser,. Zouni, W. Senger, Cynoteril photosystem II t 2.9 Å resolution nd the role of quinones, lipids, hnnels nd hloride, Nt. Strut. Mol. Biol. 16 (2009) [4] J.W. Murry, K. Mghloui, J. Krgul, N. Ishid, T.-L. Li,.W. Rutherford, M. Sugiur,. Bouss, J. Brer, X-ry rystllogrphy identifies two hloride inding sites in the oxygen evolving entre of photosystem II, Energy Environ. Si. 1 (2008) [5] K. Kwkmi, Y. Umen, N. Kmiy, J.-R. Shen, Lotion of hloride nd its possile funtions in oxygen-evolving photosystem II reveled y X-ry rystllogrphy, Pro. Ntl. d. Si. U. S (2009) [6] B.. Diner, F. Rppport, Struture, dynmis, nd energetis of the primry photohemistry of photosystem II of oxygeni photosynthesis, nnu. Rev. Plnt Biol. 53 (2002) [7] M.L. Groot, N.P. Pwlowiz, L.J. vn Wilderen, J. Breton, I.H. vn Stokkum, R. vn Grondelle, Initil eletron donor nd eptor in isolted photosystem II retion enters identified with femtoseond mid-ir spetrosopy, Pro. Ntl. d. Si. U. S (2005)

10 128. Bouss et l. / Biohimi et Biophysi t 1807 (2011) [8].R. Holzwrth, M.G. Muller, M. Reus, M. Nowzyk, J. Snder, M. Rogner, Kinetis nd mehnism of eletron trnsfer in intt photosystem II nd in the isolted retion enter: pheophytin is the primry eletron eptor, Pro. Ntl. d. Si. U. S (2006) [9] F. Rppport, B.. Diner, Primry photohemistry nd energetis leding to the oxidtion of the Mn 4 C luster nd to the evolution of moleulr oxygen in Photosystem II, Coord. Chem. Rev. 252 (2008) [10] G. Renger, T. Renger, Photosystem II: the mhinery of photosyntheti wter splitting, Photosynth. Res. 98 (2008) [11] H. Du, I. Zhriev, Priniples, effiieny, nd lueprint hrter of solr-energy onversion in photosyntheti wter oxidtion,. Chem. Res. 42 (2009) [12] Y. Kto, M. Sugiur,. Od, T. Wtne, Spetroeletrohemil determintion of the redox potentil of pheophytin, the primry eletron eptor in photosystem II, Pro. Ntl. d. Si. U. S (2009) [13].R. Crofts, C.. Wright, The eletrohemil domin of photosynthesis, Biohim. Biophys. t 726 (1983) [14] B. Bouges-Boquet, Eletron trnsfer etween the two photosystems in spinh hloroplsts, Biohim. Biophys. t 14 (1973) [15] B.R. Velthuys, J. mesz, Chrge umultion t the reduing side of system 2 of photosynthesis, Biohim. Biophys. t 333 (1974) [16] C.. Wright, Oxidtion-redution physil hemistry of the eptor quinone omplex in teril photosyntheti retion enters: evidene for new model of heriide tivity, Isr. J. Chem. 21 (1981) [17] B.R. Velthuys, Eletron-dependent ompetition etween plstoquinone nd inhiitors for inding to photosystem II, FEBS Lett. 126 (1981) [18] B. Kok, B. Forush, M. MGloin, Coopertion of hrges in photosyntheti O 2 evolution. I. liner four step mehnism, Photohem. Photoiol. 11 (1970) [19] P. Joliot, B. Kok. Oxygen evolution in photosynthesis; in: Bioenergetis of Photosynthesis (Govindjee, ed.), demi Press, New York. 1975, pp [20] Y. Nkmur, T. Kneko, S. Sto, M. Ikeuhi, H. Ktoh, S. Ssmoto,. Wtne, M. Iriguhi, K. Kwshim, T. Kimur, Y. Kishid, C. Kiyokw, M. Kohr, M. Mtsumoto,. Mtsuno, N. Nkzki, S. Shimpo, M. Sugimoto, C. Tkeuhi, M. Ymd, S. Tt, Complete genome struture of the thermophili ynoterium Thermosynehoous elongtus BP-1, DN Res. 9 (2002) [21] P.B. Kos, Z. Dek, O. Cheregi, I. Vss, Differentil regultion of ps nd psd gene expression, nd the role of the different D1 protein opies in the ynoterium Thermosynehoous elongtus BP-1, Biohim. Biophys. t 1777 (2008) [22] B. Loll, M. Broser, P.B. Kos, J. Kern, J. Biesidk, I. Vss, W. Senger,. Zouni, Modeling of vrint opies of suunit D1 in the struture of photosystem II from Thermosynehoous elongtus, Biol. Chem. 389 (2008) [23] M. Sugiur, Y. Kto, R. Tkhshi, H. Suzuki, T. Wtne, T. Noguhi, F. Rppport,. Bouss, Energetis in photosystem II from Thermosynehoous elongtus with D1 protein enoded y either the ps1 or ps3 gene, Biohim. Biophys. t 1797 (2010) [24] S..P. Merry, P.J. Nixon, L.M.C. Brter, M.J. Shilstr, G. Porter, J. Brer, J.R. Durrnt, D. Klug, Modultion of quntum yield of primry rdil pir formtion in photosystem II y site direted mutgenesis ffeting rdil tions nd nions, Biohemistry 37 (1998) [25] F. Rppport, M. Guergov-Kurs, P.J. Nixon, B.. Diner, J. Lvergne, Kinetis nd pthwys of hrge reomintion in photosystem II, Biohemistry 41 (2002) [26]. Cuni, L. Xiong, R.T. Syre, F. Rppport, J. Lvergne, Modifition of the pheophytin midpoint potentil in photosystem II: modultion of the quntum yield of hrge seprtion nd of hrge reomintion pthwys, Phys. Chem. Chem. Phys. 6 (2004) [27] K. Cser, I. Vss, Rditive nd non-rditive hrge reomintion pthwys in photosystem II studied y thermoluminesene nd hlorophyll fluoresene in the ynoterium Synehoystis 6803, Biohim. Biophys. t 1767 (2007) [28]. Krieger-Liszky,.W. Rutherford, Influene of heriide inding on the redox potentil of the quinone eptor in photosystem-ii. Relevne to photodmge nd phytotoxiity, Biohemistry 37 (1998) [29]. Krieger-Liszky,.W. Rutherford, Heriide-indued oxidtive stress in photosystem II, Trends Biohem. Si. 26 (2001) [30] P. Joliot,. Joliot, Comprtive-study of the fluoresene yield nd of the C550 sorption hnge t room-temperture, Biohim. Biophys. t 546 (1979) [31] P. Joliot,. Joliot, photosystem-ii eletron-eptor whih is not plstoquinone, FEBS Lett. 134 (1981) [32] M. Sugiur,. Bouss, T. Noguhi, F. Rppport, Influene of histidine-198 of the D1 suunit on the properties of the primry eletron donor, P 680, of photosystem II in Thermosynehoous elongtus, Biohim. Biophys. t 1777 (2008) [33] M. Sugiur, Y. Inoue, Highly purified thermo-stle oxygen-evolving photosystem II ore omplex from the thermophili ynoterium Synehoous elongtus hving His-tgged CP43, Plnt Cell Physiol. 40 (1999) [34] J.L. Hughes, N. Cox,.W. Rutherford, E. Krusz, T.L. Li,. Bouss, M. Sugiur, D1 protein vrints in photosystem II from Thermosynehoous elongtus studied y low temperture optil spetrosopy, Biohim. Biophys. t 1797 (2010) [35] M. Sugiur, F. Rppport, K. Brettel, T. Noguhi,.W. Rutherford,. Bouss, Sitedireted mutgenesis of Thermosynehoous elongtus photosystem II: the O 2 evolving enzyme lking the redox tive tyrosine D, Biohemistry 43 (2004) [36] D. Bel, F. Rppport, P. Joliot, new high-sensitivity 10-ns time-resolution spetrophotometri tehnique dpted to in vivo nlysis of the photosyntheti pprtus, Rev. Si. Instrum. 70 (1999) [37] S. Un,. Bouss, M. Sugiur, Chrteriztion of the tyrosine-z rdil nd its environment in the spin-oupled S 2 Tyr Z stte of Photosystem II from Thermosynehoous elongtus, Biohemistry 46 (2007) [38] M. Ronel,. Bouss, J.L. Zurit, H. Bottin, M. Sugiur, D. Kirilovsky, J.-M. Orteg, Redox properties of the photosystem II ytohromes 559 nd 550 in the ynoterium Thermosynehoous elongtus, J. Biol. Inorg. Chem. 8 (2003) [39] C.. Kerfeld, M.R. Swy, H. Bottin, K.T. Trn, M. Sugiur, D. Csio,. Desois, T.-O. Yetes, D. Kirilovsky,. Bouss, Struturl nd EPR hrteriztion of the solule form of ytohrome -550 nd of the psv2 gene produt from the ynoterium Thermosynehoous elongtus, Plnt Cell Physiol. 44 (2003) [40] M. Sugiur, S. Hrd, T. Mne, H. Hyshi, Y. Kshino,. Bouss, Ps30 ontriutes to struturlly stilise the photosystem II omplex in the thermophili ynoterium Thermosynehoous elongtus, Biohim. Biophys. t 1797 (2010) [41] J.-L. Zimmermnn,.W. Rutherford, Photoredutnt-indued oxidtion of Fe 2+ in the eletron-eptor omplex of photosystem II, Biohim. Biophys. t 851 (1986) [42] V. Petroules, B.. Diner, Light-indued oxidtion of the eptor-side Fe(II) of photosystem-ii y exogenous quinones ting through the QB inding-site.1. Quinones, kinetis nd ph-dependene, Biohim. Biophys. t 893 (1987) [43] B.. Diner, V. Petroules, Light-indued oxidtion of the eptor-side Fe(II) of photosystem II y exogenous quinones ting through the QB inding site. 2. Blokge y inhiitors nd their effets on the Fe(III) EPR spetr, Biohim. Biophys. t 893 (1987) [44] J. Lvergne, Mode of tion of 3-(3, 4-dihlorophenyl)-1, 1-dimethylure - evidene tht the inhiitor ompetes with plstoquinone for inding to ommon site on the eptor side of Photosystem-II, Biohim. Biophys. t 682 (1982) [45] C. Fufezn, C.X. Zhng,. Krieger-Liszky,.W. Rutherford, Seondry quinone in photosystem II of Thermosynehoous elongtus: semiquinone-iron EPR signls nd temperture dependene of eletron trnsfer, Biohemistry 44 (2005) [46] J. Kern, B. Loll, C. Luneerg, D. DiFiore, J. Biesidk, K.F. Irrgng,. Zouni, Purifition, hrteristion nd rystllistion of photosystem II from Thermosynehoous elongtus ultivted in new type of photoioretor, Biohim. Biophys. t 1706 (2005) [47] R. Krivnek, J. Kern,. Zouni, H. Du, M. Humnn, Spre quinones in the Q B vity of rystllized photosystem II of Thermosynehoous elongtus, Biohim. Biophys. t 1767 (2007) [48] J.M. Peloquin, R.D. Britt, EPR/ENDOR hrteriztion of the physil nd eletroni struture of the OEC Mn luster, Biohim. Biophys. t 1503 (2001) [49] M.-F. Chrlot,. Bouss, G. Blondin, Towrds spin oupling model for the Mn 4 luster in photosystem II, Biohim. Biophys. t 1708 (2005) [50] L.V. Kulik, B. Epel, W. Luitz, J. Messinger, Eletroni struture of the Mn 4 O x C luster in the S 0 nd S 2 sttes of the oxygen-evolving omplex of photosystem II sed on pulse Mn-55-ENDOR nd EPR Spetrosopy, J. m. Chem. So. 129 (2007) [51].R. Corrie, J.H.. Nugent, M.C.W. Evns, Identifition of EPR signls from the Q Q B nd Q B in photosystem II from Phormidium lminosum, Biohim. Biophys. t 1057 (1991) [52].W. Rutherford, J.-L. Zimmermnn, new EPR signl ttriuted to the primry plstosemiquinone eptor in photosystem II, Biohim. Biophys. t 767 (1984) [53] J.P. Dekker, H.J. vngorkom, M. Brok, L. Ouwehnd, Optil hrteriztion of photosystem-ii eletron-donors, Biohim. Biophys. t 764 (1984) [54] G.H. Shtz, H.J. vngorkom, soreny differene spetr upon hrge-trnsfer to seondry donors nd eptors in photosystem-ii, Biohim. Biophys. t 810 (1985) [55] J. Lvergne, sorption hnges of photosystem-ii donors nd eptors in lgl ells, FEBS Lett. 173 (1984) [56] J. Lvergne, Improved UV-visile spetr of the S-trnsitions in the photosyntheti oxygen-evolving system, Biohim. Biophys. t 1060 (1991) [57] G. Renger, B. Hnsum, Studies on the deonvolution of flsh-indued sorption hnges into the differene spetr of individul redox steps within the wteroxidizing enzyme-system, Photosynth. Res. 16 (1988) [58] D.B. Knff, D.I. rnon, Spetrl evidene for new photoretive omponent of oxygenevolving system in photosynthesis, Pro. Ntl. d. Si. U. S.. 63 (1969) [59] H.J. vn Gorkom, Identifition of redued primry eletron-eptor of photosystem-ii s ound semiquinone nion, Biohim. Biophys. t 347 (1974) [60] D.H. Stewrt, P.J. Nixon, B.. Diner, G.W. Brudvig, ssignment of the Qy sorne nds of photosystem II hromophores y low-temperture optil spetrosopy of wild-type nd mutnt retion entres, Biohemistry 39 (2000) [61] B.. Diner, Dependene of detivtion retions of photosystem-ii on redox stte of plstoquinone pool- vried under neroi onditions equiliri on eptor side of photosystem-ii, Biohim. Biophys. t 460 (1977) [62] P.L. Dutton, D.F. Wilson, Redox potentiometry in mitohondril nd photosyntheti ioenergetis, Biohim. Biophys. t 346 (1974) [63] J. lri, Y. Pierre, D. Piot, J. Lvergne, F. Rppport, Spetrl nd redox hrteriztion of the heme (i) of the ytohrome (6)f omplex, Pro. Ntl. d. Si. U. S (2005)