Detergent effect on cytochrome b559 electron paramagnetic resonance signals in the photosystem II reaction centre

DETERGENT EFFECT ON CYTOCHROME b559 ELECTRON PARAMAGNETIC RESONANCE SIGNALS IN THE PHOTOSYSTEM II REACTION CENTRE. Y uela, I.1*, Ga cía-Rubio, I.2, Roncel, M.3, Ma ínez, J.I.2, Rami o, M.V.1, O ega, J.M.3, Alonso, P.J.2 and Pico el, R1. 1 Es ación Expe imen al de Aula Dei, Consejo Supe io de In es igaciones Cien í icas, Apdo. 202, E-50080-Za agoza, Spain. 2 Ins i u o de Ciencia de Ma e iales de A agón, Consejo Supe io de In es igaciones Cien í icas-Uni e sidad de Za agoza, Plaza San F ancisco s/n, E-50009-Za agoza, Spain. 3 Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa-Consejo Supe io de In es igaciones Cien í icas, Ame ico Vespucio s/n, E-41092-Se illa, Spain. Co esponding au ho : I. Y uela; [email p o ec ed], Fax: +34 976 716145, phone: +34 976 716058 Keywo ds: cy och ome b559, de e gen , elec on pa amagne ic esonance, pho osys em II, eac ion cen e, edox po en ial. 1 ABSTRACT De e gen e ec on Cy och ome b559 om spinach pho osys em II was s udied by elec on pa amagne ic esonance (EPR) spec oscopy in D1-D2-Cy b559 complex p epa a ions. Va ious n-dodecyl-β-D-mal oside concen a ions om 0 o 0.2% (w/ ) we e used o s abilise he D1-D2-Cy b559 complexes. Low spin heme EPR spec a we e ob ained bu gz ea u e posi ions changed depending on de e gen condi ions. Redox po en iome ic i a ions showed a unique edox po en ial cy och ome b559 o m (E ‘m = +123-150 mV) in all he D1-D2-Cy b559 complex p epa a ions indica ing ha de e gen does no a ec his p ope y o he p o ein in hose condi ions. Simila e ec on Cy och ome b559 EPR spec um was obse ed in mo e in ac pho osys em II p epa a ions independen ly o hei agg ega ion s a e. This inding indica es ha changes due o de e gen could be a common phenomenon in pho osys em II complexes. Resul s a e discussed in e ms o he en i onmen each de e gen p o ides o he p o ein. 2 1. INTRODUCTION Cy och ome (Cy ) b559 is an in eg al componen o pho osys em (PS) II eac ion cen e (RC). I s p esence is c i ical o he biogenesis and s able assembly o he PSII RC, bu i is no in ol ed in he p ima y elec on anspo in PSII. D1-D2-Cy b559 complexes wi h he minimum polypep ide composi ion ha a e able o pe o m e icien ligh -induced p ima y cha ge sepa a ion s ill bound Cy b559.1 Ha sh ea men s a e equi ed o sepa a e Cy b559 om his complex 2,3 and he igh ela ionship wi h he D1/D2 he e odime ha binds he essen ial co ac o s o PSII p ima y pho ochemis y sugges s an essen ial ole o Cy b559. Howe e , despi e many s udies pe o med du ing he las decades4-6 he exac unc ion(s) o his hemop o ein is s ill unclea . Cy och ome b559 consis s o wo small polypep ides, he α (9 kDa) and β (4.5 kDa) subuni s wi h a ansmemb ane α-helical domain and he e odime ic s uc u e.6,7 Two his idine esidues wi hin he hyd ophobic domain o each polypep ide ac as ligands o heme i on. One o he mos in iguing p ope ies o Cy b559 is he ema kable a iabili y o he midpoin edox po en ial (E’m) o he heme g oup. In i s na u al memb ane en i onmen i exhibi s a labile high-po en ial (HP, E’m ≈ +380 mV) and a s able low- po en ial (LP, E’m ≈ +20 - +200 mV) o m. An in e media e po en ial (IP) was also epo ed in chlo oplas s, hylakoids and in ac PSII memb anes.6,8-12 Heme i on o Cy b559 in PSII RC is known o display he ypical low-spin elec on pa amagne ic esonance (EPR) signals gx ≈ 1.5, gy ≈ 2.3 and gz ≈ 3.0. Howe e , sligh ly di e en gz alues o Cy b559 ha e been obse ed depending on he p epa a ion and ex en o pu i ica ion o samples.6 These changes in EPR spec a we e connec ed in he pas wi h changes be ween di e en edox o ms o Cy b559. Indeed, i has been sugges ed ha HP and LP o ms o Cy b559 can be dis inguished by hei EPR spec a. The HP o m o Cy b559 was associa ed o a gz a ound 3.01-3.08 based on EPR measu emen s in chlo oplas s and PSII memb anes wi h high con en o his o m. On he o he hand, LP Cy b559 o m was ela ed o a gz in he 2.93-3.04 ange. 3 The gz alue o 2.93 was measu ed in he isola ed Cy b559.6,13-17 Ne e heless, a s aigh o wa d ela ionship be ween edox po en ial and EPR signal is no clea . O he ac o s ha we e sugges ed o in luence he EPR gz posi ion we e he pu i ica ion deg ee o he sample and he hyd ophobici y o heme en i onmen .6,14 In o de o elucida e ac o s con olling EPR signal, we s udy he e in de ail EPR spec a o oxidised LP Cy b559 in he D1-D2-Cy b559 complex and in mo e in ac PSII p epa a ions. Ou esul s clea ly demons a e ha de e gen s modi y he EPR spec um o Cy b559 and sugges ha EPR signal a ia ions and midpoin edox po en ial could no be di ec ly ela ed. The way de e gen s a ec EPR pa ame e s is discussed in e ms o a mo e gene al concep o hyd ophobici y e e ed in.14 4 2. MATERIALS AND METHODS P epa a ion o PSII memb anes.- Highly en iched-PSII memb anes we e isola ed om ma ke spinach acco ding o Be hold e al.18 Samples we e suspended in 0.4 M suc ose, 15 mM NaCl, 5 mM MgCl2 and 50 mM 2-(N-mo pholino) e hanesul onic acid (Mes)-NaOH, pH 6.0, ozen in liquid ni ogen and s o ed a –80ºC un il use. PSII memb anes exhibi ed oxygen e olu ion a es o 520 ± 30 μmol o O2 mg Chl-1 h-1 using DCBQ as a i icial elec on accep o . P epa a ion o D1-D2-Cy b559 complexes.- A s anda d D1-D2-Cy b559 complex p epa a ion con aining six chlo ophyll (Chl) molecules pe RC was isola ed om highly pu i ied oxygen-e ol ing PSII memb anes om ma ke spinach18 acco ding o he p ocedu e o Nanba and Sa oh1 and modi ied by Mon oya e al.19 This me hod makes use o a Toyopea l TSK-DEAE column. Samples loaded in he column we e washed wi h 0.05% (w/ ) T i on X-100 un il abso bance a 417 nm was highe han ha a 435 nm. De e gen was subsequen ly exchanged by n-dodecyl-β-D-mal oside (β-DM) a di e en concen a ions anging om 0 o 0.2% (w/ ) o 0.15% (w/ ) suc ose monocap a e. De e gen eplacemen was done un il T i on X-100 abso bance a 280 nm was lowe han 0.01. Then, D1-D2-Cy b559 complexes we e elu ed wi h a linea sal g adien in he same bu e and ac ions we e collec ed a 1 ml/min. This me hod was also used wi h h ee di e en modi ica ions desc ibed in he li e a u e.20-22 The a ia ions, conce ning basically he column and washing bu e condi ions a e he ollowing: i) Toyopea l TSK-DEAE column, 1% (w/ ) T i on X-100; ii) Toyopea l TSK-DEAE column, 1% (w/ ) T i on X-100 and 1.5% (w/ ) au ine ; iii) Q- Sepha ose Fas -Flow (Pha macia) column and 0.15% (w/ ) T i on X-100. The β-DM concen a ion in he elu ion bu e was 0.1% (w/ ) o hese la e samples and de e gen eplacemen was done as explained abo e. All isola ed D1-D2-Cy b559 complex p epa a ions con ained six Chl pe RC. 5 D1-D2-Cy b559 complexes we e isola ed a di e en pH in he 5.5-7.7 ange by changing he pH o he washing bu e . Fo pH 5.5-6.5 and pH 7.0-7.7 Mes-NaOH and is(hyd oxyme hyl)aminome hane (T is)-HCl bu e s, espec i ely, we e used. Desal ed D1-D2-Cy b559 complex p epa a ions we e p epa ed by a 2 h dialysis agains he sal ee elu ion bu e ee o sal using a 30,000 kDa cu -o dialysis ube (Spec apo ). All isola ion p ocedu es we e done in da kness in a cooled chambe a 4 ºC. Samples we e hen ozen in liquid N2 and s o ed a –80 ºC. Pigmen composi ion o isola ed D1-D2-Cy b559 complex p epa a ions was de e mined as desc ibed in Eijckelho and Dekke .23 Cy och ome b559 con en was calcula ed om he di hioni e- educed minus e icyanide-oxidised abso p ion di e ence spec a using an ex inc ion coe icien o 21.0 mM-1cm-1 a 559 nm.1 P epa a ion o PSII co e complexes.- PSII co e complex samples we e p epa ed ollowing he me hod desc ibed in24 wi h some modi ica ions. An ion exchange Toyopea l TSK-DEAE column was used. The column was washed a 2 ml/min o 2 h and subsequen ly he co e complexes we e elu ed wi h a linea sal g adien in 50 mM Mes-NaOH, pH 6.5 wi h 0.03% (w/ ) T i on X-100. Addi ionally, PSII co e complexes we e elu ed om he column a e de e gen exchange wi h 0.1% (w/ ) β-DM in he same bu e . De e gen exchange was done un il T i on X-100 abso bance a 280 nm was lowe han 0.01. Pigmen ed ac ions we e concen a ed in Cen ipep (Amicon) ubes. Isola ion o monome ic and dime ic PSII RC and co e complexes.- . Monome ic and dime ic complexes we e isola ed by suc ose densi y g adien cen i uga ion. To do ha , PSII RC we e suspended in 50 mM Mes-NaOH, pH 6.5 o 50 mM T is-HCl, pH 7.2 and 0.1% β-DM (w/ ).25 PSII co es we e suspended in 25 mM Mes-NaOH, pH 6.5, 10 mM NaCl, 5 mM CaCl2 and 10 mM NaHCO3 and incuba ed wi h β-DM o a inal concen a ion o 1.25% (w/ ).26 The solubilised PSII RC and PSII co e samples we e homogenised, loaded on o a eshly p epa ed 0.1-1.0 M suc ose g adien and 6 cen i uged a 90,000xg in a Beckman SW41 swing-ou o o o e nigh and 75,000xg in a Beckman SW28 swing-ou o o o 22 h, espec i ely. The suc ose g adien bu e composi ion was 10 mM NaCl, 50 mM Mes-NaOH, pH 6.5 o 50 mM T is-HCl, pH 7.2 and 0.1% (w/ ) β-DM o PSII RC samples and 10 mM NaCl, 5 mM CaCl2, 25 mM Mes- NaOH, pH 6.5, and 0.03% (w/ ) β-DM o PSII co es. The chlo ophyll- ich ac ions we e hen emo ed om he suc ose g adien s, ozen in liquid ni ogen and s o ed a - 80 ºC. Po en iome ic edox i a ions.- Po en iome ic edox i a ions we e ca ied ou unde a gon a 12 ºC using D1-D2-Cy b559 complex samples (5 μM Chl) in 50 mM Mes-NaOH, pH 6.5, by ollowing he abso bance changes a 559 minus 570 nm induced by sequen ial addi ion o aliquo s o 0.1 M sodium di hioni e. The measu emen s we e pe o med in an Aminco DW-2000 UV-Vis spec opho ome e using he dual wa eleng h mode. Samples we e p e iously oxidised wi h 25 μM po assium e icyanide. The edox po en ial in he eac ion cell we e simul aneously measu ed wi h a po en iome e (Me h om He isau, Swi ze land) p o ided wi h a combined P -Ag/AgCl mic oelec ode (C ison Ins umen s, Spain) p e iously calib a ed agains a sa u a ed solu ion o quinhyd one (E’m, pH 7, +280 mV a 20 ºC). In addi ion o e icyanide (E’m, pH 7, +430 mV) he ollowing edox media o s we e used: 10 μM 1,4-benzoquinone (E’m, pH 7, +280 mV), 20 μM 2,3,5,6- e ame hyl-p-phenylendiamine (E’m, pH 7, +240 mV), 20 μM 1,2- naph hoquinone (E’m, pH 7, +145 mV), 2.5 μM N-me hyl-phenazonium me hosul a e (E’m, pH 7, +80 mV), 10 μM N-me hyl-phenazonium e hosul a e (E’m, pH 7, +55 mV) and 20 μM e ame hyl-p-benzoquinone (E’m, pH 7, +5 mV). EPR measu emen s.- Samples we e concen a ed (0.5 – 1.2 mM Chl) in Cen ipep-30 and Cen icon-30 (Amicon) ubes o EPR measu emen s. Con inuous wa e EPR spec a we e eco ded wi h a B uke ESP380E spec ome e wo king a he X-band 7 ( equency abou 9.6 GHz). Typical measu emen s we e achie ed a 8 K wi h 1.46 μW mic owa e powe (which ensu es no sa u a ion e ec s on he signal) and 1 mT o modula ion ampli ude. 8 3. RESULTS E ec o n-dodecyl- β -D-mal oside on Cy b559 EPR signal. The in luence o β-DM on Cy b559 EPR spec um was obse ed in a s anda d D1-D2-Cy b559 complex p epa a ion con aining six Chl pe RC. D1-D2-Cy b559 complexes we e isola ed in he p esence o a ious β-DM concen a ions anging om 0 o 0.2% (w/ ) ( o de ails see Ma e ials and Me hods). No pigmen and polypep ide composi ion a ied among samples a e de e gen ea men s. All samples displayed a ypical low spin heme EPR signal, wi h p incipal alues o he g enso being a ound gz ≈ 3.0, gy ≈ 2.3 and gx ≈ 1.5.6 Al hough all spec a we e simila , some di e ences we e de ec ed in hei gz ea u es, wi h no changes a gx and gy posi ions. Following Taylo ’s model27 i is expec ed ha gy alue emains nea ly una ec ed when a small shi in gz alue occu s. Besides, conside ing ha he gx ea u e is e y b oad, i is no possible o de ec small changes in i as hose obse ed o gz. In e media e ield ea u e in he EPR spec a s ay a gy = 2.26 o all samples and, when he high ield ea u e is de ec ed, a gx alue o 1.53 is ob ained. Figu e 1 shows he de e gen e ec on he low ield ea u e in EPR spec a o Cy b559. The D1-D2-Cy b559 complex sample suspended in he p esence o 0.1% (w/ ) β-DM displays a gz ea u e wi h a maximum a 2.98 and a ull wid h a hal maximum (FWHM) o 13 mT (Fig. 1A,a). On he o he hand, D1-D2-Cy b559 complex samples in he p esence o lowe β-DM concen a ion (0.03% (w/ ) β-DM) display a gz ea u e wi h a maximum a 2.93 ha seems o be asymme ic showing a smoo he dec ease owa ds lowe ields and FWHM o 15 mT (Fig. 1A,b). An EPR signal wi h simila gz maximum alue has been epo ed in he li e a u e o D1-D2-Cy b559 complexes isola ed ollowing he same p ocedu e.28 The dependence o gz posi ion on β-DM concen a ion is shown in Fig. 1B. The concen a ion a which gz peak shi s (0.03-0.06% (w/ )) is abo e he c i ical micelle concen a ion (c.m.c.) o β-DM.29 A c.m.c. de e gen monome molecules sel - 9 ABBREVIATIONS Chl, chlo ophyll; c.m.c, c i ical micelle concen a ion; Cy , cy och ome; D1 and D2, co e polypep ides o he pho osys em II eac ion cen e; DCBQ, 2,6-dichlo obenzoquinone; E’m, midpoin po en ial; EPR, elec on pa amagne ic esonance; FWHM, ull wid h a hal maximum; HP, high po en ial; IP, in e media e po en ial; LP, low po en ial; Mes, 2- (N-mo pholino)e hanesul onic acid; PS, pho osys em; RC, eac ion cen e; T is, is(hyd oxyme hyl)aminome hane. REFERENCES [1] O. Nanba and K. Sa oh, Isola ion o a pho osys em II eac ion cen e consis ing o D1 and D2 polypep ides and cy och ome b559, P oc. Na l. Acad. Sci. USA, 1987, 84, 109-112. [2] X.S. Tang, K. Fushimi and K. Sa oh, D1-D2 complex o he pho osys em II eac ion cen e om spinach. Isola ion and pa ial cha ac e iza ion, FEBS Le ., 1990, 273, 257-260. [3] W.R. Widge , W.A. 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Vännga d, The in luence o pH on he EPR and edox p ope ies o cy och ome c oxidase in de e gen solu ion and in phospholipid esicles, Biochim. Biophys. Ac a, 1979, 545, 205-214. [40] M.A. Noo de mee , G.A. Veldink and J.F.G. Vliegen ha , Spec oscopic s udies on he ac i e si e o hyd ope oxide lyase; he in luence o de e gen s on i s con o ma ion, FEBS Le ., 2001, 489, 229-232. 20 FIGURE LEGENDS Figu e 1. (A) Low ield egion o EPR spec a o oxidised Cy b559 heme in D1-D2-Cy b559 complex samples elu ed om he column in he p esence o a) 0.1% (w/ ) o β-DM; b) 0.03% (w/ ) o β-DM; c) 0.05% (w/ ) o T i on X-100; d) 0.15% (w/ ) suc ose monocap a e. (B) Dependence o he Cy b559 EPR gz alue on β-DM concen a ion. EPR condi ions: empe a u e, 8K; mic owa e powe , 1.46 μW. O he expe imen al condi ions a e desc ibed in Ma e ials and Me hods. Figu e 2. Low ield egion o EPR spec a o oxidised Cy b559 heme in D1-D2-Cy b559 complexes: a) sample elu ed om he column in he p esence o 0.03% (w/ ) T i on X-100; b) he same RC sample in (a) subsequen ly ea ed wi h 0.1% (w/ ) β-DM h ough a DEAE-Toyopea l TSK-650S column o eplace T i on X- 100; c) sample elu ed om he column in he p esence o 0.1% (w/ ) β-DM a e de e gen exchange; d) he same RC sample in (c) a e emo ing β-DM de e gen . EPR condi ions: empe a u e, 8K; mic owa e powe , 1.46 μW. O he expe imen al condi ions a e desc ibed in Ma e ials and Me hods. Figu e 3. Po en iome ic edox i a ions o Cy b559 heme in D1-D2-Cy b559 complex p epa a ions a pH 6.5 in he p esence o 0.03% (w/ ) β-DM concen a ion (μ); 0.1% (w/ ) β-DM (F); 0.15% (w/ ) suc ose monocap a e (Ì). Expe imen al condi ions a e desc ibed in Ma e ials and Me hods. Figu e 4. EPR spec a a he gz egion o he oxidised Cy b559 heme in PSII co e complex sample in he p esence o a) 0.03% (w/ ) T i on X-100; b) 0.1% (w/ ) β-DM EPR condi ions: empe a u e, 8K; mic owa e powe , 1.46 μW. O he expe imen al condi ions a e desc ibed in Ma e ials and Me hods. 21 22 200 220 240 260 280 b c d a Magne ic Field (mT) A 0.0 0.1 0.2 2.90 2.93 2.96 2.99 g z alue % (w/ ) β -DM Fig. 1 23 200 220 240 260 280 d c b a Magne ic Field (mT) Fig. 2 24 -100 0 100 200 300 0 25 50 75 100 % Reduced Cy b559 Ambien edox po en ial (mV) Fig. 3 200 220 240 260 280 b a Magne ic Field (mT) Fig. 4 25