Anabaena sp. PCC 7119 Flavodoxin as Electron Carrier from Photosystem I to Ferredoxin-NADP+Reductase: ROLE OF TRP57 AND TYR94

Anabaena sp. PCC 7119 Fla odoxin as Elec on Ca ie om Pho osys em I o Fe edoxin-NADP ⴙ Reduc ase ROLE OF TRP 57 AND TYR 94 * Recei ed o publica ion, Decembe 21, 2001, and in e ised o m, Ap il 10, 2002 Published, JBC Pape s in P ess, Ap il 11, 2002, DOI 10.1074/jbc.M112258200 Jose´ L. Casaus‡, Jose´ A. Na a o§, Manuel He a´s§, Anabel Los ao‡, Miguel A. De la Rosa§, Ca los Go´mez-Mo eno‡, Ja ie Sancho‡, and Milag os Medina‡¶ F om he ‡Depa amen o de Bioquı´mica y Biologı´a Molecula y Celula , Facul ad de Ciencias, Uni e sidad de Za agoza, Za agoza 50009 and §Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Uni e sidad de Se illa-CSIC, CIC Isla de la Ca uja, Ame´ ico Vespucio s/n, Se illa 41092, Spain The in luence o he amino acid esidues sandwiching he la in ing in la odoxin (Fld) om he cyanobac e- ium Anabaena sp. PCC 7119 in complex o ma ion and elec on ans e (ET) wi h i s na u al pa ne s, pho o- sys em I (PSI) and e edoxin-NADP ⴙ educ ase (FNR), was examined in mu an s o he key esidues T p 57 and Ty 94 . The mu an s’ abili y o o m complexes wi h ei- he FNR o PSI is simila o ha o wild- ype Fld. How- e e , some o he mu an s exhibi al e ed kine ic p op- e ies in hei ET p ocesses ha can be explained in e ms o al e ed la in accessibili y and/o he mody- namic pa ame e s. The mos no iceable al e a ion is p oduced upon eplacemen o Ty 94 by alanine. In his mu an , he p ocesses ha in ol e he ans e o one elec on om ei he PSI o FNR a e clea ly accele a ed, which migh be a ibu able o a la ge accessibili y o he la in o he educ an . Howe e , when he opposi e ET low is analyzed wi h FNR, he educed Y94A mu an ans e s elec ons o FNR sligh ly mo e slowly han wild ype. This can be explained he modynamically om a dec ease in d i ing o ce due o he signi ican shi o 137 mV in he educ ion po en ial alue o he semiquinone/hyd oquinone couple (E 1 ) o Y94A, ela i e o wild ype (Los ao, A., Go´ mez-Mo eno, C., Mayhew, S. G., and Sancho, J. (1997) Biochemis y 36, 14334–14344). The beha io o he es o he mu an s can be explained in he same way. O e all, ou da a indica e ha T p 57 and Ty 94 do no play any ac i e ole in la odoxin edox e- ac ions p o iding a pa h o he elec ons bu a e a he in ol ed in se ing an app op ia e s uc u al and elec- onic en i onmen ha modula es in i o ET om PSI o FNR while p o iding a igh FMN binding. Fla odoxins a e small ␣ / ␤ la op o eins in ol ed in ET 1 e- ac ions in mic oo ganisms and ce ain algae. They can be syn- hesized cons i u i ely o induced by a lack o i on in he cul u e medium (1, 2). The p incipal ea u e o la odoxins is ha hey con ain a nonco alen ly bound low po en ial la in co ac o (FMN) ha con e s edox p ope ies o he p o ein. The binding o FMN o la odoxin conside ably inc eases he s abili y o he co ac o semiquinone s a e by al e ing i s e- duc ion po en ials. Typically, he midpoin po en ial o he quinone/semiquinone couple, E 2 , is inc eased, whe eas ha o he semiquinone/hyd oquinone couple, E 1 , is dec eased. This allows la odoxin o beha e as a one-elec on ans e cen e ha cycles in i o be ween he semiquinone and he ully educed o ms (1, 3). The h ee-dimensional s uc u es o se e al la odoxins a e known (4–10). In mos la odoxins, he isoalloxazine ing ( he edox-ac i e moie y o FMN) is s acked be ween wo a oma ic esidues. One o hem is a widely conse ed y osine ha makes ex ensi e con ac s wi h he isoalloxazine, whe eas he o he a oma ic esidue is usually a yp ophan ha in e ac s mainly wi h he wo me hyl g oups o he isoalloxazine (11). The p oximi y o he wo a oma ic esidues o he la in ing makes hem in e es ing candida es o play a ole in modula ing he educ ion po en ials o la odoxin and, he e o e, modula - ing he p o ein’s abili y o play speci ic unc ions in he elec on ans e chains whe e i is in ol ed. The in luence o hese a oma ic esidues on FMN binding, la odoxin educ ion po- en ials, and elec on spin densi y dis ibu ion o he semiqui- none s a e has been s udied by si e-di ec ed mu agenesis using la odoxins om di e en sou ces (11–14), and s uc u es o some o hese mu an s a e a ailable (15). Howe e , o he bes o ou knowledge, he in luence o hese a oma ic esidues on la odoxin ET eac ions has no been analyzed. In Anabaena Fld, he unc ionali y o si e-di ec ed mu an s wi h emo al o su ace nega i e side chains has been epo ed, no only wi h i s physiological pa ne s (PSI and FNR) bu also wi h o he ET p o eins (16, 17). The in oduced mu a ions exe ed only li le e ec s on he eac ions wi h FNR and PSI, whe eas hey showed la ge e ec s on he nonphysiological eac ions wi h cy och ome cand cy och ome P450. In Anabaena sp. PCC 7119 Fld, he la in-sandwiching es- idues a e T p 57 and Ty 94 ( he only side chains in con ac wi h he la in ing) (9). P e ious cha ac e iza ion o se e al Anabaena sp. PCC 7119 Fld mu an s a posi ions 57 and 94 has * This wo k was suppo ed by Comisio´n In e minis e ial de Ciencia y Tecnologı´a G an BIO2000-1259 ( o C. G.-M.), CONSI⫹D, Dipu acio´n Gene al de A ago´n, G an P006/2000 ( o M. M.), Comisio´n In e minis- e ial de Ciencia y Tecnologı´a G an BQU2001-2520 ( o M. M.), Di ec- cio´n Gene al de Es udios Supe io es G an PB97-1027 and G an BMC 2001-2522 ( o J. S.), Di eccio´n Gene al de In es igacio´n G an BMC2000-0444 ( o M. A. R.), Eu opean Union Ne wo ks ERB-FM- RXCT98-0218 and HPRN-CT1999-00095 ( o M. A. R.), and Jun a de Andalucı´a Plan Andaluz de In es igacio´n G an CVI-0198 ( o M. A. R.). 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: Dep . de Bioquı´mica y Biologı´a Molecula y Celula , Facul ad de Ciencias, Uni e sidad de Za agoza, Za agoza 50009, Spain. Tel.: 34-976-762476; Fax: 34-976- 762123; E-mail: [email p o ec ed]. 1 The abb e ia ions used a e: ET, elec on ans e ; dR , 5-deaza i- bo la in; dR H䡠, semiquinone o m o dR ; Fld, la odoxin; Fld ox , Fld in he oxidized s a e; Fld d , Fld in he educed s a e; Fld sq , Fld in he semiquinone s a e; FNR, e edoxin-NADP ⫹ educ ase; FNR ox , FNR in he oxidized s a e; FNR d , FNR in he educed s a e; FNR sq , FNR in he semiquinone s a e; PSI, pho osys em I; WT, wild- ype; T icine, N-[2-hyd oxy-1,1-bis(hyd oxyme hyl)e hyl]glycine. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 25, Issue o June 21, pp. 22338–22344, 2002 © 2002 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 g22338 This is an Open Access a icle unde he CC BY license. shown ha hese a oma ic esidues, in pa icula Ty 94 , in lu- ence he co ac o educ ion po en ials and i s abso p ion spec- um. In addi ion, measu emen o he apo la odoxin-FMN complex binding ene gies showed ha bo h Ty 94 and T p 57 s eng hen he in e ac ion o apo la odoxin wi h FMN in he di e en edox s a es and a e esponsible o se ing he cha - ac e is ic educ ion po en ials exhibi ed by his p o ein (11). In con as , i has been shown ha eplacemen o Ty 94 and T p 57 only e y sligh ly in luences he elec on spin densi y dis ibu ion in he la odoxin semiquinone s a e (14). In he p esen s udy, we epo he unc ional cha ac e iza ion o Anabaena sp. PCC 7119 Fld mu an s ob ained by eplacemen o T p 57 and Ty 94 by o he a oma ic esidues as well as by alanine and leucine (11). Complex o ma ion and ET be ween PSI and Fld and be ween Fld and FNR ha e been s udied o he WT and mu a ed o ms o Fld in o de o analyze he in ol emen o Ty 94 and T p 57 in hese p ocesses. So a , none o he h ee-dimensional s uc u es o he mu an s he e cha ac- e ized is a ailable. Howe e , ecen ly, he s uc u es o wo Fld mu an s om Desul o ib io ulga is a he equi alen posi ion o Ty 94 om Anabaena Fld, Y98H and Y98W, ha e been epo ed (15). Since p ecise main-chain and side-chain supe posi ion is obse ed be ween he s uc u es o WT Flds om bo h species, specially a ound he la in ing, he unc ional beha io o he Anabaena mu an s is discussed based on he s uc u al ea u es obse ed o hese wo D. ulga is mu an s uc u es. EXPERIMENTAL PROCEDURES Biological Ma e ial—Fld mu an s om Anabaena sp. PCC 7119 we e p epa ed by oligonucleo ide-di ec ed mu a ion o he Fld gene, o e p o- duced in Esche ichia coli, and pu i ied as p e iously desc ibed (10, 11). Anabaena sp. PCC 7119 WT FNR was pu i ied om E. coli isop opyl- ␤ -D- hiogalac oside-induced cul u es ha had been p e iously ans- o med wi h he p c99a ec o con aining he FNR gene om Anabaena sp. PCC 7119, as p e iously desc ibed (18). UV- isible spec- a and SDS-PAGE elec opho esis we e used as pu i y c i e ia. PSI pa icles om Anabaena sp. PCC 7119 we e ob ained by ␤ -dodecyl mal oside solubiliza ion as desc ibed by Ro¨gne e al. (19) and modi ied by He a´se al. (20). 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 (21). Chlo ophyll concen a ion was de e mined acco ding o A non (22). The chlo ophyll/P700 a io o he esul ing PSI p epa a ions was 140:1. The same ba ches o PSI and la op o eins we e used h oughou his s udy. Spec al Analysis—UV- isible spec a we e eco ded on a KON- TRON U ikon 942 spec opho ome e . Dissocia ion cons an s, binding ene gies, and changes in ex inc ion coe icien s o he complexes be- ween WT FNR ox and he di e en Fld ox a ian s we e ob ained by di e en ial spec oscopy as p e iously desc ibed (18). The expe imen s we e pe o med on solu ions con aining ⬃20 ␮ MFNR in 4 mMpo as- sium phospha e, 1 mMEDTA bu e (pH 7.0) a 25 ⫾1°C, o which aliquo s o he di e en Fld o ms we e added. Dissocia ion cons an s and di e en ial ex inc ion coe icien s o he complexes we e ob ained by i ing he expe imen al da a o he heo e ical equa ion o a 1:1 s oichiome ic complex by means o nonlinea eg essions. S eady-s a e Enzyma ic Assays—The NADPH-dependen cy och ome c educ ase ac i i y o FNR was de e mined using he di e en Fld mu an s as he elec on ca ie om FNR o cy och ome c(18, 23). To calcula e he FNR K m alues o each Fld a ian , he s anda d eac ion mix u es con ained 50 ␮ MNADPH, 2.5 nMFNR, 0.75 mMcy och ome c (Sigma), and di e en concen a ions o he co esponding Fld mu an . All measu emen s we e ca ied ou a 25 ⫾1°Cin50m MT is/HCl, pH 8.0. S opped- low Kine ic Measu emen s—ET eac ions be ween FNR and Fld we e s udied by s opped low unde anae obic condi ions in an Applied Pho ophysics SX17.MV spec opho ome e in e aced wi h an Aco n 5000 compu e , using he SX.18MV so wa e o Applied Pho o- physics as p e iously desc ibed (18). The appa en obse ed a e con- s an s (k app ) we e calcula ed by i ing he expe imen al da a o mono- o biexponen ial kine ics. Samples we e made anae obic (in specially designed onome e s, which i ed he s opped- low appa a us) by suc- cessi e e acua ion and O 2 - ee a gon lushing. Reduced samples o FNR and Fld o s opped low we e p epa ed by pho o educ ion wi h 5-dR as desc ibed (18). All eac ions we e ca ied ou in 4 mMpo assium phos- pha e, 1 mMEDTA, pH 7.0, a 13 ⫾1°C and ollowed a 600 nm, he wa eleng h a which semiquinone o ma ion o bo h la op o eins can be ollowed. FNR and Fld we e mixed a 1:1 a ios wi h inal p o ein concen a ions anging om 7 o 9 ␮ M. Each kine ic ace was he a e age o 4–10 independen measu emen s. E o s in he de e mina- ion o he appa en obse ed a e cons an alues (k app ) we e o ⫾10%. Lase Flash Abso p ion Spec oscopy—The lase lash sys em was based on a PTI PL-2300 ni ogen lase (emission wa eleng h, 337 nm; lash du a ion, 0.6 ns; ou pu ene gy, 1.4 mJ). The lase beam was a enua ed by 80% wi h calib a ed neu al densi y il e s. Kine ic aces o lash-induced abso bance changes a 580 nm we e eco ded using a Nicole 450 digi al oscilloscope. The analysis ligh sou ce was a 100-wa ungs en-halogen lamp, which passed h ough an O iel mono- ch oma o (model 77250, bandwid h 10 nm). The de ec ion pho omul- iplie (Hamama su R928) was p o ec ed om ac inic ligh by a second monoch oma o (Jobin-Y on; model H-20, bandwid h 1 nm). The wa e- leng h o 580 nm was chosen o moni o Fld semiquinone o ma ion wi h he lowes possible con ibu ion o PSI o he abso bance change. All o he expe imen s we e ca ied ou in a 1-cm pa h leng h cu e e a 22 ⫾1°C. Unless o he wise s a ed, he s anda d eac ion mix u e con ained, in a inal olume o 1 ml, 20 mMT icine/KOH, pH 7.5, 0.03% ␤ -dodecyl mal oside, an amoun o PSI-en iched pa icles equi alen o 35 ␮ g o chlo ophyll ml ⫺1 , 0.1 ␮ Mphenazine me hosul a e, 2 mMMgCl 2 , 2m Msodium asco ba e, and Fld a he indica ed concen a ion. Asco - ba e and phenazine me hosul a e addi ion ensu es ha PSI is o ally e educed be ween lashes. Each kine ic ace was he a e age o 40–50 independen measu emen s wi h 30-s spacing be ween lashes. Sam- ples we e p o ec ed om ac inic ligh be ween lashes by an elec onic shu e synch onized wi h he lase igge . Fo mos expe imen s, he es ima ed e o in he obse ed a e cons an s was less han 20%, based on ep oducibili y and signal- o-noise a ios. Da a collec ion was as p e iously desc ibed (24). Exponen ial analyses o he oscilloscope aces we e pe o med using he Ma qua d me hod wi h he so wa e de ised by P. Se´ i (Saclay, F ance). Kine ic analyses we e ca ied ou acco ding o he wo-s ep eac ion mechanism p e iously p oposed (24, 25). E o s in he es ima ed alues o cons an s o complex associa ion (K a ) and elec on ans e (k e ) we e o ⫾15 and ⫾5%, espec i ely. The pu a i e p o ona ion a e cons an o Fld semiquinone (k p o ) a e e- duc ion by dR H䡠was de e mined by ollowing he abso bance dec ease and inc ease a 465 and 580 nm, espec i ely (26). RESULTS S eady-s a e Kine ics Analysis o he Di e en Fld Mu an s— The ca aly ic p ope ies o FNR o he la odoxin-media ed NADPH-dependen cy och ome c educ ase assay ha e been de e mined unde s eady-s a e condi ions using WT and mu- a ed la odoxins. This ac i i y in ol es, in addi ion o FNR educ ion by he py idine nucleo ide, he in e ac ion and ET be ween FNR and Fld and he inal educ ion o cy och ome c by he semiquinone o m o Fld (18). As shown in Table I, mos Fld mu an s yield k ca alues wi hin a ac o o 2 o WT, bu signi ican di e ences a e obse ed when Y94A o Y94W a e used. The alue o k ca wi h Y94A inc eases 5- old ela i e o TABLE I Midpoin educ ion po en ials o he di e en Flds (da a om Re . 11) and s eady-s a e kine ics pa ame e s o WT FNR in he NADPH- dependen cy och ome c educ ase ac i i y using WT and mu an Flds as elec on ca ie Fld Reduc ion Po en ials a S eady-s a e kine ic pa ame e s E 2 E 1 k ca K m k ca /K m mV s ⫺1 mMmM ⫺1 s ⫺1 WT ⫺212 ⫺436 43 ⫾142⫾1 1.0 ⫾0.1 Y94A ⫺203 ⫺299 222 ⫾720⫾512⫾3 Y94F ⫺186 ⫺436 24 ⫾448⫾8 0.5 ⫾0.2 Y94W ⫺182 ⫺462 ⬍5 b ND b ND W57F ⫺152 ⫺432 37 ⫾339⫾4 1.0 ⫾0.1 W57L ⫺212 ⫺407 20 ⫾321⫾5 1.0 ⫾0.5 W57A ⫺173 ⫺417 18 ⫾212⫾4 1.5 ⫾0.9 W57Y ⫺139 ⫺447 36 ⫾427⫾6 1.4 ⫾0.5 a Da a om Re . 11. b Only an es ima ion o k ca was possible due o he e y low amoun o eac ion obse ed, which was independen o Fld concen a ion. A oma ic Residues in Fla in-media ed Elec on T ans e 22339 WT, whe eas Y94W displays li le ac i i y and an es ima ed k ca alue a leas 7- old lowe han WT. Unlike WT Fld, which is much less e icien han e edoxin in his assay, he Y94A Fld mu an (k ca ⫽222 ⫾7s ⫺1 ) is an ET media o as e icien as he i on-sul u p o ein (k ca ⫽200 ⫾10 s ⫺1 (18)). FNR K m alues o mos Fld mu an s we e wi hin a ac o o 2 o WT (Table I). Only W57A showed a signi ican dec ease in K m (4- old). These da a sugges ha he a ini y o he FNR䡠Fld complex is ha dly modi ied by he mu a ions. The K m o Y94W could no be de e mined due o he lack o ac i i y obse ed in he assay. When he ca aly ic e iciency (k ca /K m ) o FNR was de e - mined using di e en Fld a ian s in he cy och ome cassay, i u ned ou ha mos Flds lead o an FNR ca aly ic e iciency simila o ha o WT. In con as , Y94A appea ed o be 1 o de o magni ude mo e e icien han WT, and Y94W was almos non eac i e. The highe e iciency o Y94A e lec s he much as e ET p ocess, whe eas he low e iciency o Y94W could be due ei he o a much slowe ET p ocess o o he o ma ion o a nonp oduc i e in e ac ion be ween Fld and ei he FNR o cy- och ome c. In e ac ion o Fld ox Va ian s wi h FNR ox —To in es iga e he e ec o he Fld mu a ions a posi ions 57 and 94 on i s in e - ac ion wi h FNR, di e ence abso p ion spec oscopy was used o di ec ly e alua e he dissocia ion cons an s o he oxidized complexes (Fig. 1). The di e en ial spec um o he WT Fld ox 䡠FNR ox complex ha has been p oposed o a ise om al e a ion o he la in en i onmen s upon Fld䡠FNR associa ion shows abso p ion maxima a ound 390 and 465 nm (18). Simila spec al pe u ba ions we e de ec ed o all o he Fld mu an s s udied he e upon mixing wi h FNR ox . I is wo h no ing he simila shape o he di e en ial spec a ob ained wi h he di e en Fld mu an s, wi h bands a ound 390 and 464–480 nm wi h he same ela i e in ensi y as o he WT Fld complex (Fig. 1) despi e he ac ha some Fld mu an s (i.e. Y94W and, especially, Y94F) p esen unusual la in abso p ion spec a (Fig. 1D) (11). This sugges s ha he Fld ox 䡠FNR ox di e ence spec um is mainly due o al e a ion o he FAD abso p ion in FNR and indica es ha he o ien a ion o he wo p o eins in he complex is no al e ed by he mu a ions he e s udied. Acco dingly, all Fld ox mu an s showed simila K d alues o he WT complex (Table II). Mo eo e , he ⌬ ⑀ alues o ansi ion band I a e no a om he alue ob ained o he WT Fld complex, wi h he maximal changes obse ed o Y94A and Y94W, whose ⌬ ⑀ alue a e ⬃2- old la ge . This inc emen ed ⌬ ⑀ could be ela ed wi h he al e a ions desc ibed in he abso p- ion spec a o hese mu an s (11). Taken oge he , hese da a indica e ha T p 57 and Ty 94 exe li le in luence on he o ien a ion and s eng h o he FNR ox 䡠Fld ox associa ion. Fas Kine ic Analysis o he In e ac ion and ET P ocess be- ween FNR and Fld Mu an s as S udied by S opped Flow—To u he in es iga e he ole o Fld T p 57 and Ty 94 in associa- ion and ET o FNR, anae obic s opped- low kine ic expe i- men s we e ca ied ou . The eac ions be ween he di e en edox s a es o Fld and FNR we e analyzed by ollowing semiquinone o ma ion a 600 nm by mixing equimolecula amoun s o bo h p o eins, each in a di e en edox s a e, ei he oxidized o educed. When FNR ox was mixed wi h Fld d ,an inc ease in abso bance a 600 nm, p e iously asc ibed o o - ma ion o bo h semiquinones (18), was de ec ed o WT Fld (no shown) and all o he mu an s (Fig. 2). Fo WT, Y94F, and W57L Fld, mos o he eac ion akes place wi hin he ins u- men al dead ime (⬃3 ms) (Table III), whe eas slowe p ocesses a e obse ed o he es o he mu an s. When he eac ion o Y94A was analyzed, he kine ic aces we e bes i ed by a wo-exponen ial equa ion, wi h k app alues o 356 and 64 s ⫺1 (Fig. 2A). A simila beha io was obse ed o W57A, al hough in his case he i s p ocess akes place mainly wi hin he ins umen al dead ime (Fig. 2B, Table III). In bo h cases, he ini ial p ocess is consis en wi h he o ma ion o he wo semiquinones (Reac ion 1), ollowed by educ ion o FNR sq o he ully educed s a e by Fld d , which is s ill in he mix u e (Reac ion 2) (18). FNRox ⫹Fld d 兵- | 0 Kd 关FNRox···Fld d兴 O ¡ ke 其FNRsq ⫹Fldsq P 1 kapp1 Fld d ⫹FNRsq 兵- | 0 Kd⬘ 关FNRsq···Fld d兴 O ¡ ke ⬘ 其Fldsq ⫹FNR d P 1 kapp2 REACTIONS 1AND 2 Mo e no iceable a e he kine ics o Y94W, W57F, and W57Y (Fig. 2B). In hese mu an s, a lag phase wi h no abso bance changes is obse ed, bo h a 600 nm (Fig. 2B) and a 460 nm (no shown). This indica es ha he accumula ion o an oblig- a o y in e media e species akes place p io o ET. Mo eo e , when he in e media e accumula es and he eac ion s a s, p ocesses much slowe han ha wi h WT Fld ake place (Table III), and hey appea o occu only o a mino ex en , as de- FIG.1. Spec oscopic cha ac e iza ion o he complexes o med be ween selec ed Fld ox o ms and FNR ox .A, abso p ion spec a o WT Fld ox .B, abso p ion spec a o Y94F Fld ox .C, di e ence abso p ion spec a ob ained du ing he i a ion o WT FNR ox (17.8 ␮ M) wi h WT Fld ox .D, di e ence abso p ion spec a ob ained du ing he i a ion o WT FNR ox (16.4 ␮ M) wi h Y94F Fld ox . TABLE II Dissocia ion cons an s, di e en ial ex inc ion coe icien s, and ee ene gies o complex o ma ion o oxidized WT FNR wi h oxidized WT and mu a ed Flds Fld K d ⌬␧ 465 nm ⌬G 0 mMmM ⫺1 cm ⫺1 Kcal mol ⫺1 WT 3.6 ⫾1.0 1.5 ⫾0.2 7.4 ⫾0.1 Y94A 2.3 ⫾0.5 2.6 ⫾0.4 7.7 ⫾0.3 Y94W 6.0 ⫾1.5 3.2 ⫾0.6 7.2 ⫾0.3 Y94F 1.0 ⫾0.5 1.6 ⫾0.5 8.2 ⫾0.3 W57F 4.0 ⫾0.8 1.0 ⫾0.1 7.4 ⫾0.2 W57L 2.8 ⫾0.5 1.2 ⫾0.1 7.5 ⫾0.1 W57A 4.5 ⫾1.0 1.0 ⫾0.3 7.4 ⫾0.6 W57Y 2.2 ⫾0.8 2.0 ⫾0.6 7.7 ⫾0.3 A oma ic Residues in Fla in-media ed Elec on T ans e 22340 duced om hei low ampli udes (Fig. 2B). The ini ial lag phase could e lec a eo ganiza ion o he ini ial complex, equi ed o achie e an o ien a ion compe en o e icien ET. The e e se eac ion has also been in es iga ed. P e ious s opped- low s udies on he eac ion o FNR d wi h WT Fld ox indica ed ha his eac ion was a ela i ely slow p ocess o which wo phases we e de ec ed (18). These we e assigned o he ollowing p ocesses. Fldox ⫹FNR d 兵- | 0 Kd 关FNR d···Fldox兴 O ¡ ke 其Fldsq ⫹FNRsq P 1 kapp1 Fldox ⫹FNRsq 兵- | 0 Kd⬘ 关FNRsq···Fldox兴 O ¡ ke ⬘ 其Fldsq ⫹FNRox P 1 kapp2 REACTIONS 3AND 4 When his eac ion was pe o med using di e en Fld mu- an s, a biphasic beha io , wi h wo phases o simila ampli- ude, was de ec ed o mos mu an s (Fig. 3A), as in he WT Fld eac ion. Thus, o Y94F, Y94W, and W57L Fld, he eac ion akes place wi h k app alues wi hin a ac o o 2 o hose wi h WT Fld (Table III), al hough he k app o he second eac ion o Y94W is signi ican ly slowe . Replacemen o T p 57 ei he by phenylalanine, alanine o y osine p oduced la ge k app alues (up o a ac o o 7 o W57A in he i s p ocess; Table III). In all o hese cases, he yp ophan side-chain has been eplaced by sho e esidues. Finally, we no ice he e ec p oduced by eplacemen o Ty 94 by alanine. I s educ ion by FNR d i s o a monoexponen ial p ocess wi h a k app alue 175- old highe han ha obse ed o WT Fld (Fig. 3B, Table III), indica ing ha emo al o he a oma ic side chain a posi ion 94 p oduces an impo an e ec in he ET mechanism om FNR o Fld. The monoexponen ial beha io obse ed o his mu an migh be due ei he o an almos ull consump ion o FNR d by he i s p ocess (Reac ion 3) o o a change in he mechanism o ET om he educed FAD o he Fld FMN. Kine ic Analysis o he Lase Flash Fas Reduc ion o Fld Va ian s by PSI—P e ious s udies ha e shown ha he educ- ion o Anabaena Fld ox o he semiquinone s a e by spinach PSI pa icles can be ollowed using lase - lash abso p ion spec os- copy (27). The s udy o he semiquinone o ma ion cons i u es a use ul eac ion model o analyze he in e ac ion o ces and elec on ans e pa ame e s in ol ed in he educ ion o Fld by PSI (16, 27, 28). In he p esen wo k, we ha e analyzed he eac i i y o Anabaena Fld owa d PSI om he same species. Reduc ion o Fld mu an s by PSI pa icles ollowed monoexpo- nen ial kine ics o WT and all o he mu an s (e en a high la op o ein concen a ion). Fig. 4 ep oduces he oscilloscope aces ob ained o Y94A, W57Y, and WT Flds and shows ha he eac ion is much slowe wi h W57Y han wi h Y94A o WT. The obse ed pseudo- i s o de a e cons an s (k obs )o WTand mu an Fld educ ion by PSI depend nonlinea ly on he con- cen a ion o la op o ein and show a sa u a ion p o ile (Fig. 5). This sugges s ha a bimolecula ansien (PSI䡠Fld) complex is o med p io o ET, acco ding o he ollowing minimal wo-s ep eac ion mechanism, as desc ibed p e iously in o he PSI/Fld sys ems (16, 27, 28). FIG.2. Time cou se o he anae obic eac ions be ween WT FNR ox wi h di e en Fld d mu an s as measu ed by s opped low. The moni o ing wa eleng h was 600 nm. Final concen a ions a e gi en in pa en heses.A, eac ion o WT FNR ox (8.5 ␮ M) wi h Y94A Fld d (8.5 ␮ M) ( esidual o his i ing is shown a he bo om). B, eac ion o WT FNR ox (7.5 ␮ M) wi h W57A Fld d (7.5 ␮ M)( illed squa es; esidual o his i ing is shown a he bo om); WT FNR ox (8.1 ␮ M) wi h W57F Fld d (8.6 ␮ M)( illed iangles); WT FNR ox (8.2 ␮ M) wi h W57Y Fld d (8.6 ␮ M)( illed diamonds) and WT FNR ox (8.4 ␮ M) wi h Y94W Fld d (8.6 ␮ M) ( illed ci cles). TABLE III Kine ic pa ame e s o he eac ions o WT FNR wi h WT and mu a ed Flds as s udied by s opped low Samples we e mixed in he s opped- low ins umen a equimolecula concen a ions (⬃8 ␮ M inal concen a ion) and a he indica ed edox s a es, T a ⫽13 ⫾1°C. Reac ions we e ollowed a 600 nm. Fld FNR ox ⫹Fld d FNR d ⫹Fld ox k app1 k app2 k app1 k app2 s ⫺1 s ⫺1 WT ⬎600 a 1.8 0.8 Y94A 356 64 400.0 Y94F ⬎600 a 1.8 1.1 Y94W 54 b 0.5 0.06 W57F 35 b 12.0 1.9 W57L ⬎600 a 3.8 2.3 W57A ⬎600 a 146 14.0 0.5 W57Y 34 b 5.1 0.8 a Mos o he eac ion occu ed wi hin he ins umen al dead ime. b A lag phase is obse ed a 600 nm un il 30–50 ms; he k app was es ima ed a e his phase. FIG.3. Time cou se o he anae obic eac ions be ween WT FNR d wi h di e en Fld ox mu an s as measu ed by s opped low. The moni o ing wa eleng h was 600 nm. Final concen a ions a e gi en in pa en heses.A, eac ion o WT FNR d (8 ␮ M) wi h WT Fld ox (8.5 ␮ M)( illed diamonds); WT FNR d (8.7 ␮ M) wi h W57A Fld ox (8.2 ␮ M) (open diamonds); WT FNR d (8.7 ␮ M) wi h W57F Fld ox (9 ␮ M)( illed ci cles); WT FNR d (10 ␮ M) wi h W57Y Fld ox (9 ␮ M)( illed iangles; esidual o his i ing is shown a he bo om); WT FNR d (8.1 ␮ M) wi h W57L Fld ox (8 ␮ M)(open squa es); WT FNR d (7.8 ␮ M) wi h Y94F Fld ox (8 ␮ M)( illed squa es) and WT FNR d (8.5 ␮ M) wi h Y94W Fld ox (8.5 ␮ M) (open ci cles). B, eac ion o WT FNR d (7.5 ␮ M) wi h Y94A Fld ox (7.5 ␮ M) ( esidual o his i ing is shown a he bo om). A oma ic Residues in Fla in-media ed Elec on T ans e 22341 PSI d ⫹Fldox - | 0 Ka 关PSI d···Fldox兴 O ¡ ke PSIox ⫹Fldsq REACTION 5 in which he elec on ans e i s -o de a e cons an (k e ) can expe imen ally be in e ed om he limi ing k obs a in ini e Fld concen a ion, whe eas K a is he equilib ium cons an o he complex. Minimal alues o K a and k e , which can be es ima ed om da a like ha in Fig. 5 using he o malism de eloped by Meye e al. (25), a e shown in Table IV o WT and mu an Flds. We ha e p e iously epo ed (26) a pH-dependen a e- limi ing s ep o WT Fld educ ion by he dR adical, which was assigned o Fld semiquinone p o ona ion. The pu a i e p o ona ion a e cons an o Fld semiquinone (k p o ) a e e- duc ion by dR has hus been de e mined wi h all mu an s he ein desc ibed, bu no signi ican di e ences a e obse ed be ween mu an s and WT Fld (Table IV). F om hese indings we can in e ha he ET a e cons an de e mined a sa u a - ing Fld concen a ion (k e ) and he p oposed p o ona ion a e cons an (k p o ) co espond o wo un ela ed, independen p o- cesses. Thus, ou da a indica e ha whe eas none o he e- placemen s pe o med a posi ion 94 o 57 p omo es signi ican changes in he K a alue o he PSI d 䡠Fld ox complex unde ou s anda d condi ions, some o he mu a ions p omo e d as ic changes in he k e om PSI o Fld. Rema kably, he k e o Y94A Fld is 1 o de o magni ude highe han ha o WT, whe eas eplacemen o Ty 94 by a yp ophan p oduces a i ually non eac i e p o ein (Table IV). Replacemen o Ty 94 by phe- nylalanine has li le e ec on he eac i i y o he la op o ein, as compa ed wi h he o he mu an s a posi ion 94, as expec ed o his conse a i e change. Wi h ega d o posi ion 57, e- placemen o yp ophan wi h any smalle hyd ophobic esidue imp o es he ET om PSI o Fld. This e ec is pa icula ly ele an in W57F, whose k e is 6 imes highe han WT. The unexpec ed esul is ha eplacemen o yp ophan by y o- sine p omo es he e e se e ec , he ET a e cons an dec eas- ing 5 imes ela i e o WT. DISCUSSION T p 57 and Ty 94 , which sandwich he isoalloxazine ing in Anabaena sp. PCC 7119 Fld, ha e been shown o in luence i s educ ion po en ials and abso p ion spec a and o s eng hen he FMN apo la odoxin in e ac ion (11). In he p esen s udy, we ha e analyzed hei in luence on he Fld in e ac ion and ET by measu ing he unc ionali y o di e en Fld mu an s a posi ions 57 and 94 wi h i s wo physiological edox pa ne s, FNR and PSI. S eady-s a e and as kine ic s udies ha e been ca ied ou o analyze he eac ions o Fld wi h FNR. The K m alues ob ained o FNR, using he di e en Fld a ian s as p o ein ca ie s in FIG.5. Dependence upon Fld concen a ion o he k obs o educ ion o WT and mu a ed Fld o ms by Anabaena PSI. Ex- pe imen al condi ions we e as desc ibed unde “Expe imen al P oce- du es.”The solid lines ha e been ob ained by i ing he expe imen al poin s o he o malism de ised by Meye e al. (25). TABLE IV Kine ic pa ame e s o he educ ion o WT and mu a ed Flds by Anabaena PSI and by deaza ibo la in as s udied by lase lash pho olysis Fld Reduc ion by PSI Reduc ion by dR (k p o ) a K a k e M ⫺1 s ⫺1 s ⫺1 WT 6.0 ⫻10 5 500 450 Y94A 7.0 ⫻10 5 5500 730 Y94F 8.0 ⫻10 5 135 610 Y94W ND b ⬍100 410 W57F 3.0 ⫻10 5 2950 625 W57L 1.0 ⫻10 5 950 440 W57A 6.0 ⫻10 5 1480 580 W57Y 4.0 ⫻10 5 105 505 a The a e cons an s we e es ima ed om he Fld concen a ion- independen kine ic aces ollowed a bo h 465 and 580 nm (see “Ex- pe imen al P ocedu es”). b No de e mined, due o ex emely slow kine ics. FIG.4.Kine ic aces showing WT and mu an Flds educ ion by Anabaena PSI. Fla op o ein concen a ion was 20 ␮ M, and he pH alue was 7.5. All kine ic aces we e well i ed o single exponen ial cu es. O he condi ions we e as desc ibed unde “Expe imen al P ocedu es.” A oma ic Residues in Fla in-media ed Elec on T ans e 22342 he NADPH-dependen cy och ome c educ ase assay (Table I), as well as he da a de i ed om di e en ial spec oscopic anal- ysis o he FNR ox -Fld ox in e ac ion (Fig. 1, Table II) indica e ha he in oduced mu a ions do no p oduce any majo e ec in he s abili y o he FNR ox 䡠Fld ox complex o in he unc ion- ali y o he FNR d 䡠Fld ox complex. Howe e , some o he mu a- ions clea ly al e he ET be ween he wo p o eins. The mos no iceable al e a ion is p oduced upon eplacemen o Ty 94 by alanine. In his mu an , p ocesses in ol ing ET om FNR d o Fld ox show a es up o 5- old highe han hose ob ained wi h WT Fld when moni o ing he FNR cy och ome c educ ase ac i i y (Table I) and up o 222- old highe when ollowing he di ec eac ion by s opped low (Fig. 3B, Table III). P e ious s udies, howe e , had shown ha FNR d is appa en ly no so well sui ed o ans e ing a single elec on o WT Fld ox as i is o Fd ox (E m ⫽⫺384 mV) (29), whe eas FNR sq is much mo e e icien ( a e cons an ⬎7000 s ⫺1 (30)). These indings e eal he impo ance o he s uc u al ea u es o he p o eins pa - icipa ing in ET p ocesses o e he modynamic ac o s (18). Since, in he case o he Y94A mu an , he in oduced side chain does no p o ide an elec onic en i onmen ha can imp o e by i sel he ET p ocess, s uc u al and he mody- namic aspec s ha e o accoun o he enhancemen obse ed. The epo ed educ ion po en ials o his mu an show an E 1 alue conside ably less nega i e han ha o WT Fld (⫺299 mV e sus ⫺436 mV), indica ing ha in his mu an he en- ance o he second elec on is he modynamically a o ed, which migh allow a ull educ ion o Y94A Fld sq by FNR d o e en by FNR sq . This could explain he high k ca alue obse ed o FNR in he NADPH-dependen cy och ome c educ ase assay when using Y94A; once enough Y94A Fld d is o med, i migh cycle be ween he hyd oquinone and semiquinone s a es when ans e ing elec ons om FNR o cy och ome c, hus wo king unde a he modynamic o ce close o ha ope a ing in he Fd assay. Howe e , an enhancemen o he ET om FNR d o Fld ox should no be disca ded. To cla i y his, we ha e ollowed he FNR d /Fld ox eac ion by s opped low. The inc ease in abso bance obse ed a 600 nm (Fig. 3B) indica es a e y as accumula ion o semiquinone s a es, which is no consis en wi h a wo-elec on ans e p ocess and sugges s a single elec on ans e and accumula ion o he wo semiquinone species. The e o e, he la ge e iciency obse ed when analyz- ing he ET om FNR d o Y94A Fld ox has o be due o a as e single ET p ocess o gene a e Y94A Fld sq . Since complex o - ma ion is appa en ly no a ec ed and he E 2 alue o his mu an is simila o ha o WT Fld, s uc u al aspec s ha e o accoun o he enhancemen o he p ocess. We sugges ha eplacemen o he la ge y osine esidue by he small alanine migh help by simply making he la in co ac o mo e accessi- ble o educ ion. Consis en ly, eplacemen o y osine by yp- ophan (wi h a la ge side chain) in he Y94W mu an would make he la in co ac o less accessible, he eby explaining he lowe ET a e om FNR d o he mu an , as shown by bo h s eady-s a e and as kine ic s udies (Tables I and III). This hypo hesis is suppo ed by he s uc u es epo ed o wo mu an s om D. ulga is Fld, Y98H and Y98W, a a posi ion equi alen o ha o Ty 94 in Anabaena Fld (15). These s uc- u es show ha , al hough he o e all olding emains he same as ha o na i e Fld, he en i onmen o he isoalloxazine ing is modi ied by he in oduced mu a ions. No iceably, in bo h mu an s he 60–64 loop, (T p 60 co esponds o T p 57 in he Anabaena Fld) is displaced and esembles mo e he s uc u e o he Fld semi educed s a e han ha o he oxidized s a e. Mo eo e , he hyd ogen-bonding ne wo k and he wa e scheme a ound he FMN a e di e en , and bo h mu an s uc- u es show a sligh bending in he FMN isoalloxazine ing. In pa icula , he s uc u e o he Y98W mu an clea ly shows a la ge shielding o he la in om he sol en , especially a posi ions N-5, whe e a p o on has o en e in o de o p oduce he educed and semiquinone s a es o FMN, and C-6 o he la in ing. This s ongly sugges s ha he obse ed dec eased eac i i y o Y94W Anabaena Fld in he ET p ocesses wi h FNR is due o a dec eased accessibili y o he la in co ac o . On he o he hand, he s uc u e o he Y98H mu an om D. ulga is Fld shows less shielding o he la in ing om he sol en , wi h he benzene ing ully exposed. This lack o shielding has been ela ed wi h he no iceable E 1 shi o mo e posi i e al- ues exhibi ed by his mu an (15). A simila E 1 shi has been ound in ou Y94A mu an (11), sugges ing also a la ge acces- sibili y o he isoalloxazine ing in his mu an , as expec ed o a Ty o Ala eplacemen . Howe e , when analyzing he opposi e p ocess, educ ion o FNR by Fld a ian s, Y94A ET is hinde ed ela i e o WT Fld. This is so because he in oduced mu a ion p oduces an impo - an al e a ion o he la in E 1 alue, which is 137 mV mo e posi i e han ha o WT, making educ ion o FNR (E m ⫽⫺323 mV (29)) much less a o able om he he modynamic poin o iew. The Y94W, W57F, and W57Y Fld mu an s (Fig. 2B)do no show any majo al e a ions o he educ ion po en ials, ye hey display a lag phase and slowe and diminished p ocesses. In hese mu an s, s uc u al aspec s mus accoun o he ob- se ed lag phase, indica i e o he accumula ion o an in e me- dia e p io o ET, and sugges ing ha complex eo ganiza ion canno achie e he op imal o ien a ion in he ansi ion s a e o e icien ET. In e ac ion and ET be ween educed PSI and he di e en Flds we e analyzed by using lase lash pho olysis. Fo WT Fld, he ob ained kine ic pa ame e s a e o he same o de o mag- ni ude as hose p e iously desc ibed o he in e ac ion o his la op o ein wi h PSI om di e en sou ces (16, 28). The sim- ila associa ion cons an s ob ained wi h ei he WT Fld o he di e en mu an s o he PSI d 䡠Fld ox complex sugges ha he binding abili y o Fld o PSI is no a ec ed by he in oduced mu a ions (Table IV). As obse ed when analyzing educ ion by FNR d , educ ion o Y94A Fld by PSI p esen s a alue o k e much highe han WT. This mus , again, be due o a highe accessibili y o he la in co ac o o educ ion in he Y94A mu an . Consis en wi h his in e p e a ion, Y94W is i ually non eac i e wi h PSI (Table IV). I is no iceable ha whe eas mos eplacemen s o T p 57 wi h smalle hyd ophobic esidues op imize he elec on ans e om PSI o Fld wi h espec o WT, an impo an dec ease is obse ed o he W57Y a ian (Table IV). This inding clea ly indica es ha he hyd oxyl g oup is impai ing he ET eac ion. The a o emen ioned s udies indica e ha emo al o he a oma ic side chain in he Y94A mu an inc eases he accessi- bili y o he co ac o o he FMN one-elec on educ ion p ocess (Tables I, III, and IV), whe eas he modynamic ac o s would a o he en ance o he second elec on (11), which would p oduce accumula ion o Fld d in he cells upon ligh i adia- ion. Howe e , he he modynamic d i ing o ce would oppose ET om ully educed Fld o i s physiological subs a e, FNR. Thus, ou da a clea ly indica e ha Ty 94 is no equi ed o ET om PSI o FNR ia Fld; howe e , he occu ence in Fld o a y osine esidue a ha posi ion is necessa y o modula e he FMN E 1 alue and o a s ong FMN binding (11). Mo eo e , i p o ides a comp omise be ween e icien PSI/Fld and Fld/FNR ET. P e ious s udies sugges ed a possible ole o T p 57 in he kine ics o Fld edox eac ions (11). Howe e , ou da a indica e ha mu an s a his posi ion can accep elec ons om FNR d as e icien ly as WT (Table I and III) and much mo e e icien ly om PSI (Table IV). Mo eo e , eplacemen o T p 57 by leucine A oma ic Residues in Fla in-media ed Elec on T ans e 22343 o alanine does no p oduce majo al e a ions in he Fld abili y o educe FNR, al hough almos no educ ion o W57F o W57Y Flds is de ec ed. Ou da a, hus, indica e ha T p 57 canno be di ec ly in ol ed in he ET p ocess i sel bu a he p o ides an app op ia e s uc u al and elec onic en i onmen while coop- e a ing wi h Ty 94 in FMN binding (11). In summa y, in he ET p ocess om PSI o FNR ia Fld, he a oma ic esidues s acking he FMN co ac o appea no o be di ec ly in ol ed in he ET pa hway. Thus, in he case o Anabaena Fld, he la in en i onmen , p o ided by T p 57 and Ty 94 , migh no be he mos “p oduc i e”one o each indi id- ual p ocess, bu i ep esen s an e icien comp omise o he di e en ET s eps and allows a igh FMN binding in o de o op imize he o e all p ocess o ET om PSI o NADP ⫹ . REFERENCES 1. Mayhew, S. G., and Tollin, G. (1992) in Chemis y and Biochemis y o Fla- oenzymes, Vol. III (Mu¨lle , F., ed) pp. 389–426, CRC P ess, Inc., Boca Ra on, FL 2. Roge s, L. J. (1987) in The Cyanobac e ia (Fay, P., and Van Baalen, C., eds) pp. 35–67, Else ie Science Publishing Co., Inc., New Yo k 3. Ludwig, M. L, and Luschinsky, C. L. (1992) in Chemis y and Biochemis y o Fla oenzymes, Vol. III (Mu¨lle , F., ed) pp. 427–466, CRC P ess, Inc., Boca Ra on, FL 4. Wa enpaugh, K. D., Sieke , L. C., and Jensen, L. H. (1973) P oc. Na l. Acad. Sci. U. S. A. 70, 3857–3860 5. Bu ne , R. M., Da ling, G. D., Kendall, D. S., Lequesne, M. E., Mayhew, S. G., Smi h, W. W., and Ludwig, M. L. (1974) J. Biol. Chem. 249, 4383–4392 6. Smi h, W. W., Pa idge, K. A., Ludwig, M. L., Pe sko, G. A., Tse noglou, D., Tanaka, M., and Yasunobu, K. T. (1983) J. Mol. Biol. 165, 737–753 7. Fukuyama, K., Wakabayashi, S., Ma suba a, H., and Roge s, L. J. (1990) J. Biol. Chem. 265, 15804–15812 8. an Mie lo, C. P. M., Lijnzaad, P., Ve oo , J., Mu¨lle , F., Ve endsen, H. J., and de Vlieg, J. (1990) Eu . J. Biochem. 194, 185–198 9. Rao, S. T., Sha ie, F., Yu, C., Sa yshu , K. A., S ockman, B. J., Ma kley, J. L., and Sunda alingan, M. (1992) P o ein Sci. 1, 1413–1427 10. Genzo , C. G., Pe ales-Alco´n, A., Sancho, J., and Rome o, A. (1996) Na . S uc . Biol. 3, 329–332 11. Los ao, A., Go´mez-Mo eno, C., Mayhew, S. G., and Sancho, J. (1997) Biochem- is y 36, 14334–14344 12. Swenson, R. P., and K ey, G. D. (1994) Biochemis y 33, 8505–8514 13. Zhou, Z. M., and Swenson, R. P. (1996) Biochemis y 35, 15980–15988 14. Medina, M., Los ao, A., Sancho, J., Go´mez-Mo eno, C., Cammack, R., Alonso, P. J., and Ma ı´nez, J. (1999) Biophys. J. 77, 1712–1720 15. Reynolds, R. A., Wa , W., and Wa enpaugh, K. D. (2001) Ac a C ys allog . Sec. D57, 527–535 16. Na a o, J. A., He a´s, M., Genzo , C. G., Chedda , G., Filla , M. F., De la Rosa, M. A., Go´mez-Mo eno, C., Cheng, H., Xia, B., Chae, Y. K., Yan, H., Wong, B., S aus, N. A., Ma kley, J. L., Hu ley J. K., and Tollin, G. (1995) A ch. Biochem. Biophys. 321, 229–238 17. Jenkins, C. M., Genzo , C. G., Filla , M. F., Wa e man, M. R., and Go´mez- Mo eno, M. (1997) J. Biol. Chem. 36, 22509–22513 18. Medina, M., Ma ı´nez-Ju´l ez, M., Hu ley, J. K., Tollin, G., and Go´mez-Mo eno, C. (1998) Biochemis y 37, 2715–2728 19. Ro¨gne , M., Nixon, P. J., and Dinne , B. A. (1990) J. Biol. Chem. 265, 6189–6196 20. He a´s, M., O ega, J. M., Na a o, J. A., De la Rosa, M. A., and Bo in, H. (1994) Biochim. Biophys. Ac a 1184, 235–241 21. Ma his, P., and Se´ i , P. (1981) Is . J. Chem. 21, 316–320 22. A non, D. I. (1949) Plan Physiol. 24, 1–15 23. Shin, M. (1971) Me hods Enzymol. 23, 440–442 24. He a´s, M., Na a o, J. A., Dı´az, A., Bo in, H., and De la Rosa, M. A. (1995) Biochemis y 34, 11321–11326 25. Meye , T. E., Zhao, Z. G., Cusano ich, M. A., and Tollin, G. (1993) Biochemis y 32, 4552–4559 26. Na a o, J. A., He a´s, M., Pueyo, J. J., Medina, M., Go´mez-Mo eno, C., De la Rosa, M. A., and Tollin, G. (1994) Pho ochem. Pho obiol. 60, 231–236 27. Medina, M., He a´s, M., Na a o, J. A., De la Rosa, M. A., Go´mez-Mo eno, C., and Tollin, G. (1992) FEBS Le . 313, 239–242 28. Mu¨hlenho , U., and Se´ i , P. (1996) Biochemis y 35, 1367–1374 29. Hu ley, J. K., Webe -Main, A. N., S anko ich, M. T., Benning, M. M., Thoden, J. B., Vanhooke, J. L., Holden, H. M., Chae, J. K., Xia, B., Cheng, H., Ma kley, J. L., Ma ı´nez-Ju´l ez, M., Go´mez-Mo eno, C., Schmei s, J. L., and Tollin, G. (1997) Biochemis y 36, 11100–11117 30. Walke , M. C., Pueyo, J. J., Go´mez-Mo eno, C., and Tollin, G. (1990) A ch. Biochem. Biophys. 281, 76–83 A oma ic Residues in Fla in-media ed Elec on T ans e 22344