A Single Arginyl Residue in Plastocyanin and in Cytochrome c6 from the Cyanobacterium Anabaenasp. PCC 7119 Is Required for Efficient Reduction of Photosystem I

A Single A ginyl Residue in Plas ocyanin and in Cy och ome c 6 om he Cyanobac e ium Anabaena sp. PCC 7119 Is Requi ed o E icien Reduc ion o Pho osys em I* Recei ed o publica ion, Augus 4, 2000, and in e ised o m, Sep embe 29, 2000 Published, JBC Pape s in P ess, Sep embe 29, 2000, DOI 10.1074/jbc.M007081200 Fe nando P. Molina-He edia, Manuel He a´s, Jose´ A. Na a o, and Miguel A. De la Rosa‡ F om he Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Cen o de In es igaciones Cien ı´ icas Isla de la Ca uja, Uni e sidad de Se illa y Consejo Supe io de In es igaciones Cien ı´ icas, Ame´ ico Vespucio s/n, 41092 Se illa, Spain Posi i ely cha ged plas ocyanin om Anabaena sp. PCC 7119 was in es iga ed by si e-di ec ed mu agenesis. The eac i i y o i s mu an s owa d pho osys em I was analyzed by lase lash spec oscopy. Replacemen o a ginine a posi ion 88, which is adjacen o he coppe ligand His-87, by glu amine and, in pa icula , by glu a- ma e makes plas ocyanin educe i s a ailabili y o ans e ing elec ons o pho osys em I. Such a esidue in he coppe p o ein hus appea s o be iso unc ional wi h A g-64 (which is close o he heme g oup) in cy o- ch ome c 6 om Anabaena (Molina-He edia, F. P., Dı´az- Quin ana, A., He a´ s, M., Na a o, J. A., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 33565–33570) and Syn- echocys is (De la Ce da, B., Dı´az-Quin ana, A., Na a o, J. A., He a´ s, M., and De la Rosa, M. A. (1999) J. Biol. Chem. 274, 13292–13297). O he mu a ions conce n spe- ci ic esidues o plas ocyanin ei he a i s posi i ely cha ged eas ace (D49K, H57A, H57E, K58A, K58E, Y83A, and Y83F) o a i s no h hyd ophobic pole (L12A, K33A, and K33E). Mu a ions al e ing he su ace elec os a ic po en ial dis ibu ion allow he coppe p o ein o mod- ula e i s kine ic e iciency: he mo e posi i ely cha ged he in e ac ion si e, he highe he a e cons an . Whe eas eplacemen o Ty -83 by ei he alanine o phe- nylalanine has no e ec on he kine ics o pho osys em I educ ion, Leu-12 and Lys-33 a e essen ial o he eac- i i y o plas ocyanin. Plas ocyanin (Pc), 1 a small single coppe p o ein, and cy o- ch ome c 6 (Cy ), a monoheme p o ein, unc ion as al e na i e mobile elec on ca ie s be ween he wo memb ane complexes b 6 and pho osys em I (PSI) (c . Re s. 1–3 o e iews). The isoelec ic poin o Pc and Cy a ies widely depending on he o ganism; he wo molecules exhibi he same alue o ⬃9in he cyanobac e ium Anabaena sp. PCC 7119 (4). Thei espec i e h ee-dimensional s uc u es ha e epea - edly been sol ed using p o eins om di e en o ganisms, and hei s uc u e- unc ion ela ionships ha e been compa a i ely analyzed. In Pc, which was in es iga ed i s , wo ac i e si es we e iden i ied: si e 1 (o he so-called no h hyd ophobic pole), loca ed in a la egion a ound he coppe ligand His-87, and si e 2 (o he so-called eas ace), which is e e ed o as he acidic pa ch in euka yo ic o ganisms because i includes aspa - ic and glu amic esidues a posi ions 42–45 and 59–61 su - ounding he sol en -exposed Ty -83 (1, 2). Recen da a indi- ca e ha si e 2 is esponsible o he elec os a ic in e ac ions wi h cy och ome and PSI, whe eas si e 1, in pa icula His-87, is in ol ed in elec on ans e i sel (3, 5). We ha e ecen ly epo ed ha Cy om Synechocys is (6) and Anabaena (7) possesses wo a eas equi alen o hose o Pc: si e 1, which is a hyd ophobic egion a he edge o he heme pocke p o iding he con ac su ace o elec on ans e , and si e 2, which is a cha ged pa ch d i ing he elec os a ic mo e- men owa d i s wo memb ane-ancho ed pa ne s. Ou mu- agenesis s udies o hese wo cyanobac e ial Cy s e ealed he exis ence o a highly conse ed a ginine esidue a posi ion 64, which is loca ed on he p o ein su ace a he on ie be ween si es 1 and 2, e y close o he heme g oup, ha is essen ial o elec on ans e o PSI (6, 7). A g-64 is he only a ginine esidue in Anabaena Cy , as is A g-88 in Anabaena Pc. A g-64 in Cy could hus be he coun e pa o A g-88 in Pc. Ano he peculia i y o Pc and Cy om Anabaena, as com- pa ed wi h he p o eins om o he sou ces, is hei high iso- elec ic poin (see abo e) because o hei high lysine con en . In ac , si e 2 is posi i ely cha ged in bo h Pc and Cy , whe eas i is ypically nega i e in o he cyanobac e ia and, in pa icu- la , in highe plan s (2). Conside ing ha i is si e 2 ha mainly d i es he a ac i e mo emen o hese wo mobile me allop o eins owa d he memb ane complexes, i was o in e es o in es iga e in Anabaena Pc (as p e iously done in Cy ) how he su ace elec os a ic po en ial dis ibu ion and edox kine ics a e al e ed by mu a ions. This wo k was hus aimed a compa ing he ole o speci ic amino acids in Anabaena Pc wi h hose o i s coun e pa Cy om he same o ganism. Pc was modi ied by mu agenesis o speci ic esidues ei he a si e 1 o si e 2, wi h special a en ion being paid o A g-88. The kine ic mechanism o PSI educ ion by mu an Pcs was analyzed by lase lash abso p ion spec oscopy. EXPERIMENTAL PROCEDURES Pu i ica ion o Na i e Plas ocyanin—Pc om Anabaena sp. PCC 7119 was pu i ied as desc ibed p e iously (8), wi h he ollowing wo excep ions. i) Pc samples we e applied on o he CM-cellulose column a e oxida ion wi h po assium e icyanide, and ii) elu ion o he ad- so bed p o eins was pe o med wi h a linea g adien o 2–30 mM po assium phospha e bu e , pH 7.0, con aining 50 ␮ Mpo assium e i- cyanide. Pc concen a ion was de e mined spec opho ome ically using an abso p ion coe icien o 4.5 mM ⫺1 cm ⫺1 a 597 nm o he oxidized p o ein (9). Cons uc ion o Mu an s—The mu an pe E genes we e cons uc ed by he polyme ase chain eac ion wi h he QuickChange ki (S a - * This esea ch was suppo ed by he Di eccio´n Gene al de In es i- gacio´n Cien ı´ ica y Te´cnica (G an PB96–1381), Eu opean Union (Ne - wo ks ERB-FMRX-CT98–0218 and HPRN-CT1999–00095), and Jun a de Andalucı´a (PAI, CVI-0198). The cos s o publica ion o his a icle we e de ayed in pa by he paymen o page cha ges. This a icle mus he e o e be he eby ma ked “ad e isemen ” in acco dance wi h 18 U.S.C. Sec ion 1734 solely o indica e his ac . ‡ To whom co espondence should be add essed. Tel.: 34 954 489 506; Fax: 34 954 460 065; E-mail: [email p o ec ed]. 1 The abb e ia ions used a e: Pc, plas ocyanin; Cy , cy och ome c 6 ; PSI, pho osys em I; WT, wild- ype. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 1, Issue o Janua y 5, pp. 601–605, 2001 © 2001 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in U.S.A. This pape is a ailable on line a h p://www.jbc.o g 601 This is an Open Access a icle unde he CC BY license. agene) using oligonucleo ides o 26–38 bases, 15 ng o DNA empla es, and 16 cycles o 12 min in ex ension ime. The cons uc ion o he Anabaena pe E gene p e iously desc ibed was used as a empla e (4). The DNA agmen s we e sequenced o check he mu a ions. O he molecula biology p o ocols we e s anda d (10). P oduc ion o Recombinan P o eins and Pu i ica ion P ocedu es— T ans o med cells o Esche ichia coli MC1061 we e g own in a 40-li e e men o (Bios a C, B. B aun Bio ech) a 37 °C o 18 h unde ae obic condi ions. The e men o was illed wi h 20 li e s o s anda d Lu ia- Be ani medium (10) supplemen ed wi h 100 ␮ g/ml ampicillin and 200 ␮ MCuSO 4 . The ai low was adjus ed au oma ically o keep he con- cen a ion o dissol ed oxygen a ⬃95% o i s sa u a ion alue. The cul u e was s i ed a 250 pm. The pH alue was kep cons an a ⬃6.0 by addi ion o small amoun s o 1 NHCl. To p e en loss o he exp es- sion ec o , E. coli cells we e ans o med and immedia ely used o inocula e he eac o . Cells we e collec ed by angen ial il a ion in a Sa ocon c oss- low il a ion sys em (Sa o ius), and he pe iplasmic ac ion was ex- ac ed by eezing he cell pas e a ⫺20 °C. The ozen cell pas e was esuspended in 100 ml o deionized wa e and cen i uged. The esul - ing suspension was ex ensi ely dialyzed agains 2 mMpo assium phos- pha e, pH 7.0, wi h he excep ion o mu an s K33E, H57E, K58E, and R88E, which we e dialyzed agains 2 mMT is-HCl, pH 8.0. F om his poin on, he pu i ica ion p ocedu e o mos o he mu an s was iden- ical o ha o na i e Pc, wi h mino changes in elu ion g adien s. The mu an s K33E, H57E, and K58E we e applied on o a DEAE-cellulose column equilib a ed wi h 2 mMT is-HCl, pH 8.0. Elu ion o he ad- so bed p o eins was pe o med wi h a linea g adien o 0–100 mM NaCl in he same bu e . R88E was pu i ied by gel il a ion in a Sephadex G-50 column. In all cases, 50 ␮ Mpo assium e icyanide was added o he g adien bu e s o keep Pc oxidized. P o ein concen a ion was de e mined as desc ibed abo e. Redox Ti a ions—The edox po en ial alue o each Pc mu an was de e mined as epo ed p e iously (4, 11) by ollowing he di e en ial abso bance changes a 597 minus 500 nm. E o s in he expe imen al de e mina ions we e less han 10 mV. P epa a ion o PSI Pa icles—PSI pa icles we e isola ed om Anabaena cells by ␤ -dodecyl mal oside solubiliza ion (12, 13). The chlo- ophyll:P700 a io o he esul ing PSI p epa a ions was ⬃140:1. The P700 con en in PSI samples was calcula ed om he pho oinduced abso bance changes a 820 nm using he abso p ion coe icien o 6.5 mM ⫺1 cm ⫺1 de e mined by Ma his and Se´ i (14). Chlo ophyll concen- a ion was de e mined acco ding o he me hod o A non (15). Lase - lash Abso p ion Spec oscopy—Kine ics o lash-induced ab- so bance changes in PSI we e ollowed a 820 nm as desc ibed p e i- ously (16). The s anda d eac ion mix u e and o he expe imen al con- di ions ha e been epo ed (17). The bu e used h oughou his wo k was 20 mMT icine/KOH, pH 7.5. Da a collec ion, as well as kine ic and he modynamic analyses, we e ca ied ou as epo ed p e iously (16, 18). Appa en he modynamic pa ame e s we e es ima ed as in Dı´az e al. (19) by i ing he expe imen al da a o he equa ion o Wa kins e al. (20). Expe imen al e o s we e less han 10% o bo h he kine ic cons an s and he modynamic pa ame e s. S uc u e Simula ions—S uc u e and su ace elec os a ic po en ial dis ibu ion o Pc mu an s we e modeled using he Swiss-Pdb Viewe p og am (21). The solu ion s uc u e o educed Pc om Anabaena a iabilis (22), whose amino acid sequence exhibi s 100% iden i y wi h ha o Pc om Anabaena sp. PCC 7119, was used as a empla e. The quali y o he modeled s uc u es o each Pc mu an was es ed using he PROCHECK p og am (23). RESULTS AND DISCUSSION Se en esidues o Anabaena Pc we e chosen o be mu a ed, wo a he no h hyd ophobic pa ch and i e a he eas cha ged a ea (Fig. 1). The s e ic hind ance and na u e o in e ac ions a si e 1 we e in es iga ed by eplacing Leu-12 by Ala, and Lys-33 by Ala o Glu. The unc ional equi alence o si e 2 in Anabaena Pc and in he nega i ely cha ged coppe p o eins was analyzed by subs i u ing esidues a posi ions 49, 57, and 58; Asp-49 was eplaced by Lys, and bo h His-57 and Lys-58 we e eplaced by ei he Ala o Glu. Ty -83 was changed o Ala and Phe o check whe he Ty -83 is in ol ed in edox eac ions, a ole ha was i s p oposed by He e al. (24) bu la e disca ded by Bendall e al. (25) in o he Pcs. Ou analysis o all p ima y sequences o Pcs deposi ed a he P o ein Da a Bank wi h he BLAST sea ch p og am (26) e- ealed ha A g-88 is conse ed in cyanobac e ia bu no in highe plan s o in g een algae. The eason o such conse a- ion o A g-88 in cyanobac e ial bu no in euka yo ic Pcs was hus in es iga ed by eplacing A g-88 wi h ei he glu amine, as i is in spinach, o wi h glu ama e. Nei he he elec onic abso p ion spec um no he midpoin edox po en ial o Anabaena Pc was al e ed by he mu a ions men ioned abo e, he eby e ealing ha he coppe cen e was no dis o ed. As shown in Table I, he only excep ions we e he R88E and K33E mu an s, whose midpoin edox po en ial al- ues a e 25–30 mV lowe han ha o WT Pc. The kine ic p o ile o PSI educ ion unde s anda d condi- ions was monoexponen ial wi h all mu an s, as i is wi h WT Pc (16), bu he a e cons an s a ied g ea ly. The obse ed pseudo i s -o de a e cons an (k obs ) o PSI educ ion by any mu an is linea ly dependen on Pc concen a ion, as is he case wi h he WT molecule (Fig. 2). This inding can be in e p e ed by assuming ha he e is no o ma ion o a de ec able ansien complex be ween PSI and Pc, in ag eemen wi h a collisional kine ic model (3, 16). The bimolecula a e cons an o he o e all eac ion (k bim ) can be calcula ed unde s anda d condi ions om he linea plo s o k obs e sus Pc concen a ion. Table I shows ha mos mu an s yield k bim alues smalle han WT Pc, wi h he excep- ion o D49K, which exhibi s a highe eac i i y, and he wo mu an s a Ty -83, which a e sligh ly a ec ed. As expec ed, all mu an s o he hyd ophobic pa ch su e a signi ican dec ease in hei k bim alue. Elec os a ic mu a ions a posi ions 33, 57, and 58 cause Anabaena Pc o dec ease i s a ailabili y o educe PSI as much as i s global cha ge is made mo e nega i e. Majo changes a e ob ained wi h he A g-88 mu an s, because e- placemen o A g-88 wi h glu amine o glu ama e induces a dec ease in k bim o ⬃8 and 24 imes, espec i ely (Table I). Fig. 2 shows how ine icien he R88Q and R88E mu an s a e, e en a high concen a ions, compa ed wi h he WT molecule. Taking in o accoun he elec os a ic na u e o he in e ac- ions be ween Pc and PSI, a de ailed analysis o he e ec o ionic s eng h on k bim was pe o med. The k bim alues o WT Pc and all i s mu an s dec ease s eadily wi h inc easing NaCl concen a ion, indica ing he exis ence o a ac i e elec o- FIG.1.Space- illing model o Anabaena plas ocyanin showing he esidues modi ied by mu agenesis. The molecule is o ien ed wi h i s ypical eas ace (elec os a ic a ea a ound Ty -83; si e 2) in on , whe eas he no h hyd ophobic pa ch (o si e 1) is a he op. The mu an esidues a e depic ed in g ay, and His-87 is depic ed in black. Each esidue is iden i ied wi h wo numbe s, he i s co esponding o i s posi ion in he coppe p o ein om highe plan s and he second (be ween pa en heses) co esponding o i s place in he amino acid sequence o Anabaena plas ocyanin. Iso unc ionali y o A ginines in Plas ocyanin and Cy och ome c 6 602 s a ic in e ac ions be ween he eac ion pa ne s ha a e o e - whelmed a high ionic s eng h. The only excep ion is he R88E mu an , o which he k bim alues a e independen o ionic s eng h (see Fig. 4). The o malism de eloped by Wa kins e al. (20) acili a es he analysis o he in insic eac i i y o edox pa ne s in he absence o elec os a ic in e ac ions. By applying he Wa kins equa ion o ou da a on he ionic s eng h dependence o k bim , alues o he bimolecula a e cons an ex apola ed o in ini e ionic s eng h (k in ) can be calcula ed. As shown in Table I, all mu an s, excep L12A and R88E, exhibi k in alues simila o ha o WT Pc, indica ing ha he mino eac i i y o Pc mu- an s unde s anda d condi ions is mainly due o elec os a ic a he han hyd ophobic o s uc u al changes. In he case o L12A, howe e , he k in alue is 7- old lowe han ha o WT Pc. This sugges s ha such an isoelec ic mu a ion may indeed induce s uc u al changes hinde ing he in e ac ion be ween Pc and PSI, as p e iously obse ed in Synechocys is (17) and spinach (27). The Wa kins equa ion, howe e , canno be ap- plied as such o R88E because i s in e ac ions wi h PSI a e independen o ionic s eng h. The k bim alue o R88E a any NaCl concen a ion (1.46 ⫻10 6 M ⫺1 s ⫺1 ) is 4- old lowe han he k in alue o WT Pc (see Table I). This sugges s ha he elec os a ic change induced by mu a ion is no he only ac o a ec ing he eac i i y o R88E owa d PSI. Wo h men ioning is he inding ha he R64E mu an o Anabaena Cy exhibi s ak in alue ha is also 4- o 5- old lowe han ha o WT Cy (7). The na u e o in e ac ions be ween PSI and Pc was u he in es iga ed by pe o ming a he modynamic analysis o PSI educ ion by he coppe p o ein mu an s. In all cases, he empe a u e dependence o he obse ed a e cons an yielded linea Ey ing plo s wi h no b eakpoin s, om which he alues o he appa en ac i a ion en halpy (⌬H ‡ ), en opy (⌬S ‡ ), and ee ene gy (⌬G ‡ ) o he o e all eac ion can be calcula ed. As shown in Table I, he mos signi ican di e ence is obse ed wi h he R88E mu an , whose ee ene gy change is 8.0 kJ mol ⫺1 highe han ha o WT Pc, as expec ed om i s impai ed eac i i y owa d PSI. Such a di e ence in ⌬G ‡ is mainly due o a dec ease o 25.6 J mol ⫺1 K ⫺1 in he en opic e m. Also in e es ing is he L12A mu an , which exhibi s a ee ene gy change o 4.3 kJ mol ⫺1 highe han ha o WT Pc ha is mainly due o he dec ease in he en opic e m. The obse ed changes in ⌬G ‡ wi h he o he mu an s can be a ibu ed o shi s in he en halpic and/o en opic e ms. To elucida e whe he he lowe eac i i y o R88E can be asc ibed o easons o he han he elec os a ic change i sel , i s a ailabili y o ans e elec ons o PSI om spinach and Synechocys is was analyzed in compa ison wi h Anabaena WT Pc. The k bim alue o spinach PSI educ ion was 3.5 ⫻10 5 M ⫺1 s ⫺1 wi h R88E, o 4.3- old lowe han ha wi h Anabaena WT Pc (1.5 ⫻10 6 M ⫺1 s ⫺1 ). The k bim alue o Synechocys is PSI educ ion was 1.9 ⫻10 6 M ⫺1 s ⫺1 wi h R88E, o 15- old lowe han ha wi h Anabaena WT Pc (2.8 ⫻10 7 M ⫺1 s ⫺1 ). These indings allow us o conclude ha A g-88 is a c ucial esidue no only o long ange elec os a ic a ac ions be ween Pc and PSI bu also o elec on ans e . I should be no ed ha glu amine a posi ion 88 o spinach Pc has been eplaced wi h aspa agine, glu ama e, lysine, and y osine wi h no signi ican changes in he kine ics o PSI educ ion (27, 28). Di alen ca ions like Mg 2⫹ ha e p e iously been epo ed o be speci ically in ol ed in Pc/PSI in e ac ions in o he o gan- isms, bu no Anabaena (8). As such, an analysis o he e ec o Mg 2⫹ ca ions on he bimolecula a e cons an o PSI educ ion by WT and mu an Pcs was pe o med. The expe imen al e- sul s showed ha he h ee mu an s wi h one posi i e cha ge eplaced by a nega i e esidue (K33E, K58E, and R88E) a e he only ones al e ed by magnesium ca ions. Wi h hese mu an s he bimolecula a e cons an exhibi s highe alues wi h MgCl 2 han wi h NaCl a he same ionic s eng h (da a no shown). All hese indings can be in e p e ed by assuming no only ha he added nega i e cha ges in Pc mu an s acili a e he o ma ion o new sal b idges wi h posi i e coun e pa s in PSI (16), bu also ha he o iginal posi i e esidues in WT Pc in e ac wi h an acidic a ea in PSI. The posi i ely cha ged si e 2 in Anabaena Pc does appea o TABLE I Midpoin edox po en ial (E m ) o wild- ype and mu an plas ocyanins, as well as bimolecula a e cons an s and ac i a ion pa ame e s o he o e all eac ion o PSI educ ion by he di e en coppe p o eins Plas ocyanin E m ,pH7.0 k bim ⫻10 ⫺7 k in ⫻10 ⫺6 ⌬G ‡ ⌬H ‡ ⌬S ‡ mV M ⫺1 s ⫺1 M ⫺1 s ⫺1 kJ mol ⫺1 kJ mol ⫺1 J mol ⫺1 K ⫺1 Wild- ype 355 7.68 5.96 27.9 36.2 27.7 Mu an s a he eas ace R88E 325 0.32 ND a 35.9 36.5 2.1 R88Q 336 0.99 5.78 33.0 41.4 33.0 D49K 353 9.73 6.29 27.5 35.3 26.2 H57A 345 1.70 6.09 31.7 39.1 24.7 H57E 341 3.76 5.94 29.6 39.3 32.5 K58A 346 3.03 3.48 30.3 41.1 36.3 K58E 339 1.59 4.19 31.7 42.9 37.4 Y83A 340 4.44 6.18 29.3 44.0 49.2 Y83F 343 4.73 5.22 29.1 34.0 16.5 Mu an s a he no h pole L12A 371 1.37 0.84 32.2 31.0 ⫺4.2 K33A 355 2.55 3.75 30.7 40.2 31.9 K33E 330 1.35 3.10 32.2 41.9 32.3 a ND, no de e mined, because his mu an shows a k bim independen o ionic s eng h. FIG.2. Dependence upon plas ocyanin concen a ion o he obse ed a e cons an (k obs ) o PSI educ ion by he WT spe- cies and R88 mu an s o he coppe p o ein. Expe imen al condi- ions we e as desc ibed unde “Expe imen al P ocedu es.” Iso unc ionali y o A ginines in Plas ocyanin and Cy och ome c 6 603 play he same ole as he nega i ely cha ged si e in o he Pcs. The su ace elec os a ic po en ial dis ibu ion o Anabaena Pc was hen calcula ed o i s WT species as well as o he D49K and R88E molecules. As can be seen in Fig. 3, hei espec i e elec os a ic cha ges a si e 2 co ela e well wi h hei ela i e e iciency as elec on dono s o PSI; he mo e posi i e he local su ace cha ge, he highe he kine ic a e cons an o pho o- sys em educ ion. A simila co ela ion had p e iously been obse ed wi h a numbe o mu an s a si e 2 o Cy om Anabaena (7) and Synechocys is (6). The close simila i y be ween Pc and Cy om Anabaena was in es iga ed by compa ing he s uc u es o he wo molecules and he ela i e posi ion o c i ical su ace esidues. The h ee- dimensional s uc u es o he me allop o eins we e hus mod- elled (Fig. 4, uppe panel). Assuming ha His-87 in Pc plays he same edox ole as he heme g oup in Cy (29), he com- pa ison o he wo p o ein s uc u es makes A g-88 and Asp-49 o Pc occupy posi ions equi alen o A g-64 and Asp-72 in Cy . In addi ion, he high numbe o lysine esidues in bo h mole- cules could con ibu e o si e 2 being so posi i ely cha ged. Such a s uc u al coincidence be ween Pc and Cy om Anabaena is indeed suppo ed by he expe imen al indings conce ning he kine ic beha io o mu an s a ec ed a he a ginine and aspa a e esidues. Fig. 4 (lowe panels) shows how he k bim alues a low ionic s eng h o he D49K mu an o Pc a e signi ican ly highe han hose o WT Pc, as a e he alues o he D72K mu an o Cy compa ed wi h WT Cy . The k bim alues o R88E o Pc and R64E o Cy a e, howe e , lowe han hose o hei espec i e WT species. Also wo h no ing is he pa allel ionic s eng h dependence o k bim wi h he WT and mu an p o eins, i.e. he dec ease in k bim wi h inc easing NaCl concen a ion. Se e al possibili ies conce ning he ole o he single a ginyl esidue in bo h Cy and Pc can be de ised. The in e ac ion o hese wo me allop o eins wi h PSI should in ol e he o ma- ion o a ansien elec os a ic complex, which has in ac kine ically been de ec ed wi h Cy bu no wi h Pc, p obably due o i s low associa ion cons an . The a ginyl esidue could hus be equi ed o app op ia e o ien a ion o he edox cen- e s wi hin he ansien complex. Speci ic elec os a ic in e - ac ions o A g-88 in Pc and A g-64 in Cy wi h nega i ely cha ged g oups in PSI could be made e iden as soon as a high esolu ion s uc u e o PSI is made a ailable. In addi ion, he p oximi y o he a ginyl esidue o he p os he ic g oup in each p o ein can explain why he a ginine mu an s o bo h Pc and Cy exhibi alues o he a e cons an ex apola ed o in ini e ionic s eng h lowe han hose o he espec i e WT species. In his con ex , con o ma ional changes o A g-88 ela ed o changes in he edox s a e o Pc ha e been desc ibed ecen ly (30). To conclude, we can say ha si e 1 o Anabaena Pc is simila o si e 1 o o he Pcs, bu si e 2 is posi i ely cha ged in Anabaena, whe eas i is nega i ely cha ged in o he o ganisms. In addi ion, no only Pc bu also Cy om Anabaena con ains a single a ginine esidue be ween si es 1 and 2 ha appea s o play he same unc ion in he wo molecules. Acknowledgmen s—We hank S. Alaga a nam, A. Balme, A. Dı´az, and S. Mu doch o c i ically eading he manusc ip . REFERENCES 1. G oss, E. L. (1996) in Oxygenic Pho osyn hesis: The Ligh Reac ions (O , D. R., and Yocum, C. F., eds) pp. 413–429, Kluwe Academic Publishe s, Do d ech , The Ne he lands FIG.3. Su ace elec os a ic po en ial dis ibu ion o wild- ype plas ocyanin (WT) and i s mu an s R88E and D49K. The elec os a ic po en ial was calcula ed as indica ed unde “Expe imen al P ocedu es.” Simula ions we e pe o med assuming an ionic s eng h o 40 mMa pH 7.0. Nega i e and posi i e po en ial egions a e depic ed in ed and blue, espec i ely. P o ein molecules a e in he same o ien a- ion as in Fig. 1 (uppe ) o o a ed 180 o a ound he e ical axis (lowe ). FIG.4. Uppe panel, space- illing model o plas ocyanin and cy o- ch ome c 6 om Anabaena wi h he acidic and basic esidues in ed and blue, espec i ely. His-87 in Pc and he heme g oup in Cy a e depic ed in g een, A g-88 in Pc and A g-64 in Cy a e in ligh blue, and Asp-49 in Pc and Asp-72 in Cy a e in yellow. 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