Overoxidation of 2-cys peroxiredoxin in prokaryotes: Cyanobacterial 2-cys peroxiredoxins sensitive to oxidative stress

O e oxida ion o 2-Cys Pe oxi edoxin in P oka yo es CYANOBACTERIAL 2-CYS PEROXIREDOXINS SENSITIVE TO OXIDATIVE STRESS * □ S ⽧ Recei ed o publica ion, July 1, 2010, and in e ised o m, Augus 16, 2010 Published, JBC Pape s in P ess, Augus 24, 2010, DOI 10.1074/jbc.M110.160465 Ma ía B. Pascual, Alejand o Ma a-Cabana, F ancisco J. Flo encio, Ma ika Lindahl, and F ancisco J. Cejudo 1 F om he Ins i u o de Bioquímica Vege al y Fo osín esis, Uni e sidad de Se illa and Consejo Supe io de In es igaciones Cien í icas, 41092 Se illa, Spain In euka yo ic o ganisms, hyd ogen pe oxide has a dual e ec ; i is po en ially oxic o he cell bu also has an impo an sig- naling ac i i y. Acco ding o he p e iously p oposed loodga e hypo hesis, he signaling ac i i y o hyd ogen pe oxide in euka yo es equi es a ansien inc ease in i s concen a ion, which is due o he inac i a ion by o e oxida ion o 2-Cys pe - oxi edoxin (2-Cys P x). Sensi i i y o o e oxida ion depends on he s uc u al GGLG and YF mo i s p esen in euka yo ic 2-Cys P xs and is belie ed o be absen om p oka yo ic enzymes, hus ep esen ing a pa adoxical gain o unc ion exclusi e o euka y- o ic o ganisms. He e we show ha 2-Cys P xs om se e al p o- ka yo ic o ganisms, including cyanobac e ia, con ain he GG(L/ V/I)G and YF mo i s cha ac e is ic o sensi i e enzymes. In sea ch o he exis ence o o e oxida ion-sensi i e 2-Cys P xs in p oka yo es, we ha e analyzed he sensi i i y o o e oxida ion o 2-Cys P xs om wo cyanobac e ial s ains, Anabaena sp. PCC7120 and Synechocys is sp. PCC6803. In i o analysis o wild ype and mu an a ian s o he Anabaena 2-Cys P x showed ha his enzyme is o e oxidized a he pe oxida ic cys- eine esidue, hus cons i u ing an excep ion among p o- ka yo es. Mo eo e , he 2-Cys P x om Anabaena is eadily and e e sibly o e oxidized in i o in esponse o high ligh and hyd ogen pe oxide, showing highe sensi i i y o o e oxida ion han he Synechocys is enzyme. These cyanobac e ial s ains ha e di e en s a egies o cope wi h hyd ogen pe oxide. While Synechocys is has low con en o less sensi i e 2-Cys P x and high ca alase ac i i y, Anabaena con ains abundan and sensi- i e 2-Cys P x, bu low ca alase ac i i y, which is ema kably simila o he chlo oplas sys em. Hyd ogen pe oxide, a byp oduc o ae obic me abolism, has po en ial cy o oxic e ec s bu is also an in acellula messenge in euka yo es (1). To balance he oxic and signaling e ec s o hyd ogen pe oxide, i s in acellula concen a ion needs o be igh ly con olled. Fo ha pu pose, cells a e equipped wi h an ioxidan enzymes including pe oxi edoxins (P xs), 2 which a e hiol-based pe oxidases ha ca alyze he educ ion o hyd ogen pe oxide and alkyl hyd ope oxides by he conce ed ac ion o wo cys eine esidues (2). Al hough P xs ha e a mod- e a e ca aly ic e iciency, his is compensa ed o by he gene - ally high abundance o hese p o eins (3). Du ing ca alysis, he N- e minal cys eine, e med pe oxida ic, a acks he pe oxide and becomes ansien ly oxidized o sul enic acid, which hen eac s and o ms a disul ide b idge wi h he second cys eine esidue, e med esol ing (4). P x ecycling is in mos cases achie ed by a wo-componen sys em o med by hio edoxin and hio edoxin educ ase (NTR). Howe e , he bac e ial P x, AhpC, is educed by AhpF, a bimodula enzyme composed o an N- e minal double hio edoxin old and a C- e minal NTR domain (5). Simila ly, he plan homologue o AhpC, he chlo oplas -localized 2-Cys P x, is mos e icien ly educed by he bimodula enzyme, NTRC, composed o an NTR domain a he N e minus and a hio edoxin domain a he C e minus (6–8). The pe oxida ic cys eine esidue o 2-Cys P xs may unde go o e oxida ion om sul enic o sul inic acid (9), which p o okes he inac i a ion o he enzyme. Ini ially desc ibed as an i e- e sible p ocess, i was la e shown ha o e oxida ion may be e e sed in an ATP-dependen p ocess ca alyzed by sul i e- doxin (S x) (10, 11). The suscep ibili y o o e oxida ion has been conside ed speci ic o euka yo ic 2-Cys P xs and depends on he p esence o wo mo i s, GGLG and YF, loca ed a he C e minus o hese p o eins (12). These sequence mo i s impose a 14 Å sepa a ion o he wo ca aly ic cys eine esidues, which slows down disul ide o ma ion, and hence, he sul enic acid in e media e is occasionally o e oxidized o sul inic acid (12). Fo his eason, euka yo ic 2-Cys P xs ha e been e med “sen- si i e” in con as o he p oka yo ic enzymes, which lack he GGLG and YF mo i s, a e much less sensi i e o o e oxida ion, and a e hus e med “ obus ” (12). The dual ac ion o hyd ogen pe oxide, as cy o oxic agen and in acellula messenge , is well documen ed in euka yo es (1). In p oka yo es, howe e , hyd ogen pe oxide has no been p e- iously a ibu ed an ex ensi e signaling ac i i y wi h ew excep ions, such as ha o he ansc ip ion ac o OxyR (13, 14).Wi h heaimo explaining hisdi e en ac iono hyd ogen pe oxide as messenge in p oka yo es and euka yo es, he loodga e hypo hesis (12) p oposed ha inac i a ion o P xs by o e oxida ion ep esen s a gain o unc ion o euka yo es, which allows a apid inc ease in hyd ogen pe oxide le els and, hus,pe mi si s signaling ac i i y.Thiswould no bepossiblein p oka yo es, which a e equipped wi h obus P xs. Conse- *This wo k was suppo ed by G an s BIO2007-60644 and BFU2007-60300 om he Minis e io de Educacio´n y Ciencia (Spain) and G an s P06-CVI- 01578, BIO-182, and BIO-284 om he Jun a de Andalucia (Spain). ⽧ This a icle was selec ed as a Pape o he Week. □ S The on-line e sion o his a icle (a ailable a h p://www.jbc.o g) con ains supplemen al Tables 1 and 2 and Figs. 1 and 2. 1 To whom co espondence should be add essed: Ins i u o de Bioquímica Vege al y Fo osín esis, A da Ame´ ico Vespucio 49, 41092 Se illa, Spain, Fax: 34-954460065; E-mail: [email p o ec ed]. 2 The abb e ia ions used a e: P x, pe oxi edoxin; S x, sul i edoxin; NTR, NADPH hio edoxin educ ase; ␮ E, mic oeins eins. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 45, pp. 34485–34492, No embe 5, 2010 © 2010 by The Ame ican Socie y o Biochemis y and Molecula Biology, Inc. P in ed in he U.S.A. NOVEMBER 5, 2010•VOLUME 285•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 34485 This is an Open Access a icle unde he CC BY license. quen ly, euka yo ic P xs mus ha e e ol ed o become sensi i e o o e oxida ion. In plan s, as in o he euka yo es, hyd ogen pe oxide has an impo an signaling ac i i y (15–18). In pho osyn he ic plan cells, he chlo oplas s a e a majo sou ce o hyd ogen pe oxide (19) and con ain 2-Cys P xs suscep ible o o e oxida ion (20– 22). Mo eo e , 2-Cys P xs a e among he mos abundan chlo- oplas p o eins and a e in ol ed in hyd ogen pe oxide-depen- den signaling (23). The deg ee o o e oxida ion o chlo oplas 2-Cys P x is s ongly a ec ed by wo plas idial enzymes: NTRC, he mos e icien educ an o disul ide-bonded 2-Cys P x (21), and S x, which ca alyzes he con e sion o he o e oxidized in o he educed o m o he enzyme (22, 24). In a sequence analysis, we ound ha cyanobac e ial 2-Cys P xs ha bo GG(L/V/I)G and YF mo i s and, hence, would pos- sibly unde go o e oxida ion, unlike all p e iously epo ed p o- ka yo ic enzymes. Thus, in sea ch o sensi i e 2-Cys P xs in p oka yo ic o ganisms, we se ou o s udy 2-Cys P x sensi i i y o o e oxida ion in cyanobac e ia. To his end, we ha e ana- lyzed wo cyanobac e ial s ains: Anabaena sp. PCC 7120, which is equipped wi h NTRC and S x, and Synechocys is sp. PCC 6803, which lacks bo h NTRC and S x genes. Ou esul s show he exis ence o o e oxida ion in 2-Cys P x om hese p oka yo es in i o and in i o. The enzyme om Anabaena is conside ably mo e sensi i e han ha o Synechocys is in i o. Fu he mo e, we unco e ed wo di e en s a egies o cope wi h hyd ogen pe oxide in cyanobac e ia, and ound ha he s a egy om Anabaena is ema kably simila o he chlo o- plas sys em. EXPERIMENTAL PROCEDURES Cyanobac e ial S ains and G ow h Condi ions—Synecho- cys is sp. PCC 6803 and Anabaena sp. PCC 7120 we e g own as desc ibedp e iously(25) a a ligh in ensi yo 50 ␮ molpho ons m ⫺2 s ⫺1 . Fo expe imen s unde high ligh condi ions, cul u es a a densi y o 2.5 ␮ g o chlo ophyll/ml we e illumina ed wi h whi e ligh a an in ensi y o 800 ␮ mol pho ons m ⫺2 s ⫺1 , and he empe a u e was kep a 30 °C. Cloning, Exp ession, and Pu i ica ion o Recombinan P o- eins and Mu an Va ian s—The genes encoding 2-Cys P x o Anabaena and Synechocys is we e ampli ied om genomic DNA by PCR using gene-speci ic oligonucleo ides, which included NdeI and XhoI si es o cloning. The sequences o hese oligonucleo ides we e as ollows: Ana2CP, o wa d (5⬘-GAATTAAGCATATGTCCATCACC-3⬘) and Ana2CP e e se (5⬘-GTTCTCGAGACGCGATCGCCAAC-3⬘) o he Anabaena 2-Cys P x, and 2-Cys o wa d (5⬘-GCTACATA- TGACAGAGGTATTAAGGGTAG-3⬘) and 2-Cys e e se (5⬘- GCTACTCGAGCTAAGGTTCCGCCACTGTCTC-3⬘) o he Synechocys is 2-Cys P x. The NdeI and XhoI si es a e unde lined. The PCR p oduc s we e diges ed wi h bo h enzymes and cloned in o he exp ession ec o pET28 o p o- duce he pET-Anab2CP and pET-Syn2CP plasmids, which we e in oduced in o Esche ichia coli BL21 (DE3)-pLysS (P o- mega). Exp ession was induced by 1 mMisop opyl-L-D- hiogal- ac oside(IPTG),and he ecombinan p o eins we e pu i ied by nickel-ni ilo iace ic acid a ini y ch oma og aphy (Qiagen). Si e-di ec edmu agenesiswas pe o med by PCRusingpET- Anab2CP as he empla e DNA. In each case, mu a ions we e p oduced wi h oligonucleo ides ha included a single change (unde lined), which caused he C56S subs i u ion (5⬘-GACTT- TACCTTTGTTTCCCCCACGGAGATC-3⬘;5⬘-GATCTC- CGTGGGGGAAACAAAGGTAAAGTC-3⬘) and he C178S subs i u ion (5⬘-CCCAGATGAAGTTTCCCCTGCTGGTT- GGC-3⬘;5⬘-GCCAACCAGCAGGGGAAACTTCATCTGGG- 3⬘), espec i ely.ThePCRp oduc swe ediges ed wi h DpnI o 1 h a 37 °C o elimina e he me hyla ed empla e DNA. Non- diges ed plasmids we e he ea e used o ans o m E. coli BL21(DE3) pLysS. The co ec in oduc ion o mu a ions was e i ied by DNA sequencing. Exp ession o he mu a ed e - sions o 2-Cys P x in E. coli and pu i ica ion o he ecombinan enzymes we e ca ied ou as desc ibed o he wild ype enzymes. One- and Two-dimensional Gel Elec opho esis and Wes e n Blo Analysis—Cy osolicex ac so cyanobac e ials ainswe e p epa ed as desc ibed (25). One-dimensional SDS-PAGE was pe o med using 15% polyac ylamide gels unde non- educing o educing condi ions. Fo educing condi ions, he loading bu e included 1 mMDTT and 14 mM ␤ -me cap oe hanol. All samples con ained 10 ␮ g o p o ein and we e boiled o 5 min p io o elec opho esis. Fo isoelec ic ocusing, p o ein ex ac s we e p ecipi a ed wi h a inal concen a ion o 5% ( / ) ichlo oace ic acid, and immobilized pH g adien (pH ange 4–7) s ips o 11 cm (IPG s ips, Bio-Rad) we e applied acco d- ing o he manu ac u e ’s ecommenda ions. IPG s ips we e he ea e subjec ed o SDS-PAGE. Fo Wes e n blo analysis, p o eins esol ed on wo-dimen- sional o one-dimensional SDS-PAGE (15% ac ylamide) gels we e ans e ed o ni ocellulose memb anes and p obed wi h an ibodies aised agains he 2-Cys P x om ice (7) o he 2-Cys P x om Synechocys is, which was p oduced by immuni- za ion o abbi s wi h he pu i ied ecombinan p o ein. An i- bodies agains o e oxidized 2-Cys P x we e pu chased om LabF on ie (Seoul, Ko ea). Mass Spec ome y Analysis—Pu i ied ecombinan p o eins ea ed o no wi h hyd ogen pe oxide we e subjec ed o ma ix-assis ed lase deso p ion/ioniza ion ime-o - ligh (MALDI-TOF) mass spec ome y on an Au o lex appa a us (B uke Dal onics,B emen,Ge many). Ex e nal calib a ion was pe o med using he pep ide calib a ion s anda d (B uke Dal- onics), and he ypsin au odiges ion p oduc s o m/z alues 842.5094 and 2211.1046 we e used o in e nal calib a ion. De e mina ion o G ow h and Viabili y o Cyanobac e ial Cul u es in he P esence o Hyd ogen Pe oxide—To de e mine g ow h a es in he p esence o hyd ogen pe oxide, Anabaena andSynechocys is cul u esinexponen ialg ow hphase(3–5 ␮ g o chlo ophyll ml ⫺1 ) we e adjus ed o a densi y o 2.5 ␮ go chlo ophyll ml ⫺1 . Hyd ogen pe oxide, a inal concen a ions anging om 0 o 10 mM, was added o 30-ml aliquo s o cul- u es, which we e hen incuba ed o 24 h unde s anda d g ow h condi ions (50 ␮ mol pho ons m ⫺2 s ⫺1 ). Analysis o Hyd ogen Pe oxide Decomposi ion in Vi o—Fo analysis o hyd ogen pe oxide decomposi ion, 20-ml cul u es we e inocula ed a a concen a ion o 5 ␮ g o chlo ophyll ml ⫺1 and kep unde s anda d g ow h condi ions. The ea e , hyd o- O e oxida ion o P oka yo ic 2-Cys P x 34486 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 45•NOVEMBER 5, 2010 gen pe oxide was added o inal concen a ions o 0.5 mM, and aliquo s we e wi hd awn a a ious ime poin s o measu e- men so pe oxideconcen a ion. The amoun o hyd ogenpe - oxide emaining in he cul u es was de e mined by oxida ion o Fe 2⫹ in he p esence o xylenol o ange using he e ous oxida- ion o xylenol o ange assay (26). RESULTS Cyanobac e ial 2-Cys P xs Possess he Sequence Mo i s Cha - ac e is ic o Sensi i e Euka yo ic Enzymes—I has been p o- posed ha he suscep ibili y o o e oxida ion o 2-Cys P x, spe- ci ic o euka yo ic o ganisms, depends on he p esence o he GGLG and YF mo i s (12). A phylogene ic analysis o 2-Cys P x om di e se species dis inguished 2-Cys P xs lacking he GGLG and YF mo i s, all o which a e om p oka yo ic o gan- isms (Fig. 1 and supplemen al Table 1). Howe e , 2-Cys P xs con aining hese mo i s, hus ep esen ing sensi i e enzymes, we e ound in all euka yo ic o ganisms analyzed bu also in a numbe o p oka yo es (Fig. 1 and supplemen al Table 1). I should be no ed ha al hough he i s wo esidues o he GGLG mo i we e highly conse ed, he hi d and ou h esi- dues showed some a iabili y. As he enzymes o he cyanobac- e ial s ains he e analyzed (see below) con ained aline o isoleucine as he hi d esidue, his mo i is he ein deno ed GG(L/V/I)G. In addi ion, 2-Cys P x om bo h cyanobac e ial s ains showed he conse ed YF mo i (supplemen al Table 1). Mo eo e , he phylogene ic analysis e ealed ha plan , algal, and cyanobac e ial 2-Cys P xs a e closely ela ed and belong o he g oup o enzymes p edic ed o be sensi i e (Fig. 1, box). The 2-Cys P xs om Anabaena and Synechocys is Unde go O e oxida ion in Vi o—The abo e obse a ions aised he possibili y ha cyanobac e ial 2-Cys P xs, despi e being p o- ka yo ic enzymes, migh unde go o e oxida ion. To analyze his possibili y, he 2-Cys P xs om he cyanobac e ia Anabaena sp. PCC 7120 and Synechocys is sp. PCC 6803 we e exp essedin E. coli wi ha His ag a he N e minus andpu i ied by nickel-ni ilo iace ic acid ch oma og aphy (no shown). As anini ialapp oach,we es ed he c oss- eac i i y o an an ibody aised agains o e oxidized 2-Cys P x owa d he pu i ied cya- nobac e ial enzymes ea ed wi h DTT and hyd ogen pe oxide. Because he plan chlo oplas 2-Cys P x has been p e iously epo ed o unde go o e oxida ion, as de ec ed by hese an i- bodies (22), he pu i ied ice 2-Cys P x was included as a posi- i e con ol. Indeed, all h ee enzymes showed clea signals a e pe oxide ea men , whe eas no signals we e obse ed be o e ea men (Fig. 2). Aiming a a mo e de ailed analysis o heo e oxida ion o ap oka yo ic2-Cys P x, wegene a ed si e- di ec edmu an e sions o heenzyme om Anabaena eplac- ing ei he he pe oxida ic Cys-56 o he esol ing Cys-178 es- idues by Se (C56S and C178S mu an s, espec i ely). Pu i ied wild ype and mu an enzymes we e incuba ed wi h hyd ogen pe oxide in he p esence o DTT, and subsequen ly he o ma- ion o cys eine sul inic acid was examined by wo-dimensional gelelec opho esisandmassspec ome y.Thewild ype2-Cys P x was shi ed owa d he acidic side o he gel, which is indic- a i e o sul inic acid o ma ion (Fig. 3A). The C178S mu an lacking he esol ing cys eine exhibi ed a simila shi ; howe e , no shi was obse ed o he C56S mu an lacking he pe oxi- H.pylo i P.p o undum B.aphidicola E.canis R.sibi ica P. alcipa um T.annula a R. ub um C.pa um R.bal ica C.pneumoniae C. epidum O.sinensis Anabaena Synechocys is P.pu pu ea C. einha d ii A. haliana O.sa i a L.in e ogans T. aginalis D.discoideum C.elegans T.b ucei S.ce e isiae S.pombe C.neo o mas T.c uzi B.glab a a D.melanogas e H.sapiens E.his oly ica D. e io G. iolaceus C.pas eu ianum S. he mophilum Z.mobilis B.japonicum M. ube culosis S.usi a us C. uddii P. u iosus T.pallidum E. aecalis P.gingi alis B. ie namiensis N.a oma ici o ans S. yphimu ium B.sub ilis FIGURE 1. Phylogene ic analysis o 2-Cys P xs om euka yo ic and p o- ka yo ic o ganisms. Amino acid sequences we e ob ained om he Na ional Cen e o Bio echnology In o ma ion (NCBI) p o ein da abase, and accession numbe s o hese sequences a e p esen ed in supplemen al Table 1. The ee was ob ained by he MEGA4 package, and he neighbo -joining me hod was applied wi h 1000 boo s ap eplica ions. The scale ba indica es he e- quency o subs i u ions pe si e. Blue, p oka yo ic o ganisms; ed, euka yo ic o ganisms. The box indica es he clade o med by cyanobac e ial, algal, and plan enzymes. Anabaena ep esen s sp. PCC 7120, and Synechocys is ep e- sen s sp. PCC 6803 a e ep esen ed. H. pylo i,Helicobac e pylo i;P. p o un- dum,Pho obac e ium p o undum;B. aphidicola,Buchne a aphidicola;E. canis, Eh lichia canis;R. sibi ica,Ricke sia sibi ica;P. alcipa um,Plasmodium alcipa- um;T. annula a,Theile ia annula a;R. ub um,Rhodospi illum ub um;C. pa - um,C yp ospo idium pa um;R. bal ica,Rhodopi ellula bal ica;C. pneu- moniae,Chlamydophila pneumoniae;C. epidum,Chlo obium epidum; O. sinensis,Odon ella sinensis;P. pu pu ea,Po phy a pu pu ea;C. einha d ii, Chlamydomonas einha d ii;A. haliana,A abidopsis haliana;O. sa i a,O yza sa i a;L. in e ogans,Lep ospi a in e ogans;T. aginalis,T ichomonas agina- lis;D. discoideum,Dic yos elium discoideum;C. elegans,Caeno habdi is elegans;T. b ucei,T ypanosoma b ucei;S. ce e isiae,Saccha omyces ce e isiae; S. pombe,Schizosaccha omyces pombe;C. neo o mas,C yp ococcus neo o - mas;T. c uzi,T ypanosoma c uzi;B. glab a a,Biomphala ia glab a a;D. mela- nogas e ,D osophila melanogas e ;H. sapiens,Homo sapiens;E. his oly ica, En amoeba his oly ica;D. e io,Danio e io;C. pas eu ianum,Clos idium pas- eu ianum;S. he mophilum,Symbiobac e ium he mophilum;Z. mobilis, Zymomonas mobilis;B. japonicum,B ady hizobium japonicum;M. ube culosis, Mycobac e ium ube culosis;S. usi a us,Solibac e usi a us;C. uddii,Candida- us Ca sonella uddii;P. u iosus,Py ococcus u iosus;T. pallidum,T eponema pallidum;E. aecalis,En e ococcus aecalis;P. gingi alis,Po phy omonas gin- gi alis;B. ie namiensis,Bu kholde ia ie namiensis;N. a oma ici o ans, No osphingobium a oma ici o ans;S. yphimu ium,Salmonella yphimu ium; B. sub ilis,Bacillus sub ilis. O e oxida ion o P oka yo ic 2-Cys P x NOVEMBER 5, 2010•VOLUME 285•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 34487 da ic cys eine (Fig. 3A). Fu he mo e, mass spec ome y anal- ysis o pe oxide- ea ed C178S p o ein showed a shi o he mass o cha ge a io (m/z) o 32.3 Dal ons (Fig. 3B), consis en wi h he addi ional mass o wo oxygen a oms, no obse ed in he C56S mu an (Fig. 3C). Thus, i may be concluded ha he pe oxida ic cys eine esidue o he Anabaena 2-Cys P x unde - goes o e oxida ion o sul inic acid unde oxidizing condi ions and, he e o e, beha es as a sensi i e, euka yo ic- ype enzyme. The 2-Cys P xs om Anabaena and Synechocys is Display Di e en Sensi i i y o O e oxida ion in Vi o—To assess he physiological ele ance o 2-Cys P x o e oxida ion in cya- nobac e ia, he edox s a us o he enzyme was de e mined in i o unde s anda d g ow h condi ions and upon exposu e o cul u es o high ligh in ensi y o hyd ogen pe oxide ea - men s. Because NTRC and S x exe a g ea in luence on chlo- oplas 2-Cys P x o e oxida ion, his analysis was pe o med in wo ep esen a i e cyanobac e ial s ains, Anabaena sp. PCC 7120,which con ains NTRCand S x, andSynechocys is sp. PCC 6803, which lacks bo h enzymes (supplemen al Table 2). The wo cyanobac e ial s ains we e i s g own unde s anda d condi ions a a pho on lux densi y o 50 ␮ Em ⫺2 s ⫺1 (con ol ligh )and hen ans e ed oaligh in ensi yo 800 ␮ Em ⫺2 s ⫺1 (high ligh ). Unde con ol condi ions, 2-Cys P x was de ec ed exclusi ely in he dime ic, oxidized o m bo h in Anabaena and inSynechocys is (Fig. 4A). Thede ec ion o dime ic2-Cys P x as adouble band has been a ibu ed o he simul aneous p esence o dime s linked by wo disul ides (lowe band) o one disul ide (uppe band) (27). Fi een min ollowing he onse o high ligh , mos o he Anabaena 2-Cys P x was ound as a monome , which is indica i e o ex ensi e o e oxida ion o he enzyme (Fig. 4A). Despi e con inuous illumina ion wi h high ligh , a e 60–120 min, he monome ic o m disappea ed g adually, hence showing he e e sibili y o he p ocess. Re e sion was no inhibi ed by he addi ion o chlo amphenicol, showing ha i was independen o p o ein syn hesis. Su p isingly, he same high ligh ea men did no p oduce de ec able o e oxida ion o 2-Cys P x in Synechocys is (Fig. 4A). The an ibody used o de ec ion o o e oxida ion o he pu i ied enzymes equi ed a leas 1 ␮ g o o e oxidized cyanobac e ial 2-Cys P x (Fig. 2) and was he e o e no sensi i e enough o de ec hese p o eins in FIGURE 2. O e oxida ion o pu i ied plan and cyanobac e ial 2-Cys P x in i o.Pu i ied ecombinan 2-Cys P x (25 ␮ g o p o ein) om ice (Os), Anabaena (Ana), and Synechocys is (Syn) we e incuba ed wi h 10 mMDTT and 5m Mhyd ogen pe oxide. A he indica ed imes, he eac ion was s opped by p o ein p ecipi a ion wi h 5% ichlo oace ic acid and washed wice wi h ice- cold ace one. Samples (0.3 ␮ g o p o ein o he ice enzyme and 5 ␮ go Anabaena and Synechocys is enzymes) we e hen subjec ed o SDS-PAGE unde educing condi ions. Wes e n blo analysis was pe o med using an i- bodies agains o e oxidized 2-Cys P x. FIGURE 3. O e oxida ion o pu i ied Anabaena 2-Cys P x in i o.A, pu i- ied ecombinan wild ype and mu an e sions o Anabaena 2-Cys P x (4 ␮ M inal concen a ion) was incuba ed o 5 min in he absence o p esence o 0.5 mMhyd ogen pe oxide. The ea e , DTT was added o each sample a a inal concen a ion o 10 mM, and samples we e incuba ed o ano he 10 min and p ecipi a ed wi h 5% ichlo oace ic acid. Samples (1 ␮ g o p o ein) we e subjec ed o wo-dimensional gel elec opho esis and p o eins isualized wi h Coomassie B illian Blue. Con , con ol; ed, educed; ox, o e oxidized. Band C, MALDI-TOF analysis o mass- o-cha ge a ios (m/z) o mu an e - sions, as indica ed, o 2-Cys P x om Anabaena.Con inuous lines, con ol; dashed lines,H 2 O 2 - ea ed. In ens. [a.u.], in ensi y (a bi a y uni s). O e oxida ion o P oka yo ic 2-Cys P x 34488 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 45•NOVEMBER 5, 2010 i o, aking in o accoun hei ela i e abundance (supplemen- al Fig. 2). The e o e, o con i m he high ligh -induced o e - oxida ion o he Anabaena 2-Cys P x and i s e e sion, ex ac s we eanalyzed by wo-dimensionalgel elec opho esis (Fig.4B). These esul s show ha despi e he ac ha bo h Anabaena and Synechocys is ha e 2-Cys P x wi h he euka yo ic- ype mo i s, GG(L/V/I)G and YF, and unde go o e oxida ion in i o, he enzyme om Anabaena is mo e sensi i e in i o. To analyze u he he di e ences in sensi i i y be ween hese cyanobac e ial 2-Cys P xs, cul u es we e ea ed wi h inc easing concen a ions o exogenously added hyd ogen pe - oxide unde con ol ligh condi ions. This ea men p o oked o e oxida ion o he Synechocys is 2-Cys P x, al hough i was appa en ly less sensi i e han he Anabaena enzyme (Fig. 5). Mo eo e , mos o he dime ic o m was eco e ed in Anabaena a e 2ho u he incuba ion wi hou pe oxide, whe eas he Synechocys is 2-Cys P x, which became only pa - ially o e oxidized, e ained essen ially he same p opo ion o dime ic and monome ic o ms e en a e 4ho u he incuba- ion (Fig. 5), hus sugges ing ha o e oxida ion is no a e e s- ible p ocess in Synechocys is. Because he 2-Cys P xs om bo h Anabaena and Synecho- cys is con ain he s uc u al GG(L/V/I)G and YF mo i s and unde go o e oxida ion in i o, he di e en sensi i i y o o e - oxida ion migh be due o addi ional p ope ies o hese enzymes. We easoned ha he disul ide be ween pe oxida ic and esol ingcys einesshould p o ec he o me om eac ing wi h pe oxides. To es his possibili y, he abili y o an a i icial disul ide educ an , DTT, o educe each o hese enzymes was analyzed.The ice2-CysP xwashighlysensi i e o DTT educ- ion (Fig. 6A). O he cyanobac e ial enzymes, Anabaena 2-Cys P x was mo e p one o educ ion by DTT han he Synechocys- is enzyme. These esul s sugges ha he p opo ion educed o disul ide-bound 2-Cys P x may be a signi ican de e minan o he suscep ibili y o o e oxida ion and may unde sco e he di e en sensi i i y o he enzymes om Anabaena and Synechocys is. A sequence compa ison o 2-Cys P x om di e en sou ces e ealed a high deg ee o iden i y be ween 2-Cys P xs om plan , algae, and cyanobac e ia (supplemen al Fig. 1). The pe - cen age o iden i y be ween he 2-Cys P x om Anabaena and Synechocys is (75%) is e y simila o he iden i y be ween he homologues om Anabaena and ice (73%). Howe e , he iden i y be ween he Synechocys is and ice 2-Cys P x is lowe (63%). Mos o he di e ences be ween Synechocys is, Anabaena, and plan 2-Cys P xs a e loca ed in he icini y o he esol ing cys eine (supplemen al Fig. 1). In ag eemen wi h hese obse a ions, an ibodies aised agains he ice 2-Cys P x c oss- eac ed e icien ly wi h he Anabaena 2-Cys P x bu no wi h he Synechocys is enzyme (Fig. 6B). Con e sely, an ibodies aised agains he Synechocys is 2-Cys P x e icien ly de ec ed he enzyme om Anabaena bu no om ice (Fig. 6B). Rema kably, he 2-Cys P x om Synechocys is showed a di e - en elec opho e ical mobili y (Fig. 6B) despi e he ac ha he molecula mass o he enzyme (24.8 kDa) is simila o he Anabaena and ice enzymes (24.7 and 24.2 kDa, espec i ely). In addi ion, quan i a i e Wes e n blo analysis showed ha Synechocys is con ains a leas 20 imes less 2-Cys P x han does Anabaena (supplemen al Fig. 2). The Anabaena enzyme was es ima ed o be p esen in a p opo ion o 10 ng/ ␮ g o cy osolic p o ein, compa ible wi h he abundance o he chlo oplas FIGURE 4. E ec o high ligh on Anabaena and Synechocys is 2-Cys P x o e oxida ion in i o.A, cells g own o mid-exponen ial phase a 50 ␮ Em ⫺2 s ⫺1 we e dilu ed o a concen a ion o 2.5 ␮ g o chlo ophyll ml ⫺1 and exposed o a ligh in ensi y o 800 ␮ Em ⫺2 s ⫺1 . A e 30 min o high ligh exposu e, chlo amphenicol (200 ␮ g/ml inal concen a ion) was added o one aliquo and incuba ed o a u he 30 min (Cm). Cy osolic ex ac s o samples om Anabaena (5 ␮ g o p o ein) and Synechocys is (25 ␮ g o p o ein) ha es ed a he imes indica ed we e subjec ed o SDS-PAGE unde non- educing condi ions and Wes e n blo analysis. The o e oxidized o m o he 2-Cys P xs is dis inguished as a monome . Molecula mass ma ke s, in kDa, a e indica ed on he le .mon, monome ; dim, dime . B, ex ac s (15 ␮ g o p o ein) om Anabaena cul u es a he indica ed imes o high ligh ea men we e subjec ed o wo-dimensional gel elec opho esis and Wes e n blo . Ox, o e - oxidized; Red, educed. FIGURE 5. E ec o hyd ogen pe oxide on Anabaena and Synechocys is 2-Cys P x o e oxida ion in i o.Cul u es g own a 50 ␮ Em ⫺2 s ⫺1 o a con- cen a ion o 5 ␮ g o chlo ophyll ml ⫺1 we e incuba ed o 15 min wi h hyd o- gen pe oxide a he indica ed concen a ions. Cul u es ea ed wi h 0.5 mM hyd ogen pe oxide we e also ha es ed 2 and 4 h a e he addi ion o hyd o- gen pe oxide. Wes e n blo analysis was pe o med using an ibodies aised agains ice and Synechocys is 2-Cys P x o Anabaena and Synechocys is sam- ples, espec i ely. Molecula mass ma ke s, in kDa, a e indica ed on he le . mon, monome ; dim, dime . O e oxida ion o P oka yo ic 2-Cys P x NOVEMBER 5, 2010•VOLUME 285•NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 34489 2-Cys P x, which cons i u es abou 0.6% o he o al chlo oplas p o ein (23). These esul s emphasize he simila i ies be ween he Anabaena and chlo oplas 2-Cys P xs and he di e ences be ween hese enzymes and he Synechocys is homologue. Anabaena and Synechocys is Ha e Di e en S a egies o Cope wi h Hyd ogen Pe oxide—Nex , we examined whe he he di e en con en and sensi i i y o 2-Cys P x in he cyanobac- e ial s ains had any e ec on he esponse o hyd ogen pe ox- ide. Exponen ially g owing Synechocys is cul u es showed a highe a e o hyd ogen pe oxide decomposi ion han Anabaena (Fig.7A). Fu he mo e, he pe oxide emo al inSyn- echocys is was s ongly inhibi ed by NH 2 OH (Fig. 7A), which blocks he ac i i y o he cyanobac e ial ca alase-pe oxidase Ka G (28). In con as , pe oxide emo al by Anabaena, which lacks Ka G (29) (supplemen al Table 2), was much less a ec ed by NH 2 OH (Fig. 7A). Finally, we explo ed he consequences o hese di e en cyanobac e ial sys ems o pe oxide de oxi ica- ion wi h espec o su i al o oxida i e s ess. In ag eemen wi h he highe a e o pe oxide de oxi ica ion, Synechocys is cul u es su i ed highe concen a ions o exogenously added hyd ogen pe oxide han Anabaena (Fig. 7B). DISCUSSION Acco ding o he loodga e hypo hesis (12), he di e en ac ion o hyd ogen pe oxide in euka yo ic and p oka yo ic o ganisms depends on s uc u al cha ac e is ics o 2-Cys P xs. This means ha in con as o p oka yo ic 2-Cys P xs, euka y- o ic enzymes ha e e ol ed o become mo e sensi i e o inac i- a ion by o e oxida ion (30). Plan chlo oplas s con ain euka yo ic- ype 2-Cys P xs wi h he GG(L/V/I)G and YF mo i s, which unde go o e oxida ion o he pe oxida ic cys- eine esidue (20–22). As chlo oplas s o igina ed om oxy- genic pho oau o ophic p oka yo es simila o mode n cya- nobac e ia (31), we ha e analyzed he exis ence o 2-Cys P x sensi i e o o e oxida ion in hese p oka yo ic o ganisms. Asea ch o he GG(L/V/I)Gand YFmo i s cha ac e is ic o sensi i e 2-Cys P xs e ealed ha hese a e no exclusi e o euka yo ic o ganisms (Fig. 1 and supplemen al Table 1). Among he p oka yo es wi h pu a i e sensi i e- ype 2-Cys P x a e he cyanobac e ia, heenzymes o whicha e ela ed o hose o algae and plan s, con i ming p e ious phylogene ic analysis (32). The only excep ion is he 2-Cys P x om he cyanobac e- ium Gloeobac e iolaceus, which lacks bo h he GG(L/V/I)G and he YF mo i s (supplemen al Table 1) and appea s phylo- gene ically dis an o 2-Cys P xs om algae, plan s, and o he cyanobac e ia(Fig.1).No ably,G. iolaceus isano ganismwi h peculia p ope ies such as he absence o hylakoid mem- FIGURE 6. Compa ison o plan and cyanobac e ial 2-Cys P x. A, i a ion in i o o he pu i ied plan and cyanobac e ial 2-Cys P x wi h DTT. Samples (0.8 ␮ g o pu i ied p o ein) o 2-Cys P x om ice, Anabaena, and Synechocys is,as indica ed, we e ea ed wi h inc easing concen a ions o DTT o 10 min. P o eins we e hen subjec ed o SDS-PAGE, and bands s ained wi h Coomas- sie B illian Blue we e quan i ied wi h he ImageJ so wa e. B, immunological cha ac e iza ion o plan and cyanobac e ial 2-Cys P x. Fo Wes e n blo anal- ysis, 50 ng o he pu i ied 2-Cys P x om Synechocys is (Syn), Anabaena (Ana), o ice (Os) we e subjec ed o SDS-PAGE and p obed wi h an ibodies aised agains he ice ( ␣ -Os 2Cys P x) and Synechocys is 2-Cys P x ( ␣ -Syn 2Cys P x), as indica ed. Molecula mass ma ke s, in kDa, a e indica ed on he le . FIGURE 7. Kine ics o hyd ogen pe oxide decomposi ion in cul u es o Anabaena and Synechocys is.A, cul u es we e g own o mid-exponen ial phase, dilu ed o a concen a ion o 3.5 ␮ g o chlo ophyll ml ⫺1 and incuba ed unde no mal ligh condi ions in he p esence o absence o 0.1 mMNH 2 OH, which inhibi s he ca alase-pe oxidase ac i i y. A ime 0, hyd ogen pe oxide was added o a inal concen a ion o 0.5 mM. The emaining amoun s o hyd ogen pe oxide we e moni o ed using he e ous oxida ion o xylenol o ange assay. Œand ‚,Synechocys is in he absence (Œ) o p esence (‚)o NH 2 OH; Fand E,Anabaena in he absence (F) o p esence (E)o NH 2 OH. B, e ec o hyd ogen pe oxide on Anabaena and Synechocys is su i al. Cya- nobac e ial cul u es in mid-exponen ial g ow h phase dilu ed o a concen a- ion o 2.5 ␮ g o chlo ophyll ml ⫺1 we e incuba ed o 24 h unde s anda d ligh condi ions in he p esence o up o 10 mMhyd ogen pe oxide. O e oxida ion o P oka yo ic 2-Cys P x 34490 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285•NUMBER 45•NOVEMBER 5, 2010 b anes and, in many aspec s, di e s om o he cyanobac e ia (33). The wide dis ibu ion o 2-Cys P x p edic ed o be sensi- i e among p oka yo es sugges s ha his s uc u al ea u e appea ed ea ly du ing e olu ion. The c oss- eac i i y o he pe oxide- ea ed 2-Cys P x om Anabaena and Synechocys is wi h an an ibody agains he o e - oxidized enzyme (Fig. 2), in addi ion o he pe oxide-induced shi o he pI obse ed o he wild ype and C178S mu an o he Anabaena 2-Cys P x, bu no o he C56S mu an , com- bined wi h mass spec ome y (Fig. 3), demons a ed ha he cyanobac e ial 2-Cys P x is suscep ible o o e oxida ion o he pe oxida ic cys eine esidue. Mo eo e , expe imen s using Anabaena and Synechocys is cul u es showed ha o e oxida- ion akes place in i o in esponse o high ligh in Anabaena (Fig. 4) o o hyd ogen pe oxide ea men s bo h in Anabaena and in Synechocys is (Fig. 5). The e o e, hese esul s show ha cyanobac e ia cons i u e a p e iously un ecognized excep ion among p oka yo es in ha ing 2-Cys P x sensi i e o o e oxida- ion. In con as , 2-Cys P xs om o he p oka yo es s udied ei he a e no o e oxidized o equi e much highe hyd ogen pe oxide concen a ion o become o e oxidized in i o (12, 34–36). The analysis o 2-Cys P x o e oxida ion in i o e ealed ema kable di e ences be ween Anabaena and Synechocys is. Despi e he ac ha he 2-Cys P xs om bo h s ains possess he s uc u al de e minan s o allow o e oxida ion, he enzyme om Synechocys is was much less sensi i e (Figs. 4 and 5) and equi ed a highe concen a ion o DTT o educ ion (Fig. 6A). This la e cha ac e is ic migh explain, a leas in pa , he lowe sensi i i y o he enzyme o o e oxida ion because he disul ide-bonded o m o he 2-Cys P x should be ine o sul inic acid o ma ion. In his con ex , i should be no ed ha he high DTT concen a ion (10 mM), used o he ini ial assays o immunological de ec ion o o e oxida ion (Fig. 2), should ha e been sa u a ing, and no di e ence in o e oxida ion be ween he ice, Anabaena, and Synechocys is enzymes could be obse ed. In plan chlo oplas s, he mos e icien educ an o 2-Cys P x is NTRC (7, 8). Rema kably, in an A abidopsis NTRC knock-ou mu an , he 2-Cys P xs a e p edominan ly in he dime ic o m and display lowe le els o o e oxida ion (21), which sugges s ha he enzyme in he dime ic, oxidized o m is p o ec ed om o e oxida ion. Ano he aspec de e mining he o e oxida ion s a us o 2-Cys P x in i o is i s e e sion ca a- lyzed by S x, a chlo oplas -localized enzyme in plan s (24). I should be no ed ha Anabaena con ains genes encoding bo h NTRC and S x, whe eas Synechocys is does no (supplemen al Table 2). The e e sibili y o he 2-Cys P x o e oxida ion obse ed in Anabaena, bu no in Synechocys is, would hus be in ag eemen wi h he p esence o S x in Anabaena. The p onounced di e ences be ween Anabaena and Syn- echocys is conce ning 2-Cys P x sensi i i y o o e oxida ion indica es ha hese cyanobac e ia ha e de eloped di e en s a egies o cope wi h oxida i e s ess; al hough Synechocys is is equipped wi h an e icien mechanism o de oxi y hyd ogen pe oxide, he mechanism o Anabaena is less e icien (Fig. 7A). Despi e an ex ensi e complemen o hiol pe oxidases includ- ing a ious ypes o P x (37), he an ioxidan sys em o Synecho- cys is elies mainly on a high Ka G ca alase ac i i y, as deduced om he almos comple e inhibi ion o hyd ogen pe oxide decomposi ion by NH 2 OH (Fig. 7A) and p e ious esul s om Tichy and Ve maas (38) using a Ka G dele ion mu an . A con- sequence o such a high a e o hyd ogen pe oxide de oxi ica- ionis ha pe oxideconcen a ions in i o would a ely behigh enough o cause 2-Cys P x o e oxida ion, which is also in line wi h ou obse a ions. In con as , in Anabaena, he an ioxi- dan mechanism does no ely on ca alase (supplemen al Table 2), as con i med by he low inhibi o y e ec o NH 2 OH, bu p obably he 2-Cys P x, which is abou 20 imes mo e abundan han in Synechocys is, plays a mo e p ominen ole. These di - e en an ioxidan s a egies ha e an impo an impac on he abili y o he cyanobac e ia o su i e oxida i e s ess. The e i- cien sys em o Synechocys is allows his cyanobac e ium o ole a e highe concen a ions o hyd ogen pe oxide han Anabaena. The s a egy exempli ied by Synechocys is, based on high ca alase ac i i y and low le el o insensi i e 2-Cys P x, pu sues su i al by apid exhaus ion o he pe oxide, whe eas he s a egy o Anabaena ails o p o ide su i al. I should be no ed ha plan chlo oplas s ha e a con en o 2-Cys P x, sim- ila o ha o Anabaena (23), and he enzyme is highly sensi i e o o e oxida ion (20–22). Mo eo e , hese o ganelles a e also de oid o ca alase bu con ain NTRC and S x. Hence, ou esul s a e in ag eemen wi h he p oposal ha he endosymbi- o ic cyanobac e ial ances o , which o igina ed he plan chlo- oplas , was o he ilamen ous, he e ocys - o ming ype, such as he mode n Anabaena sp. PCC 7120 and Anabaena a iabi- lis ATCC 29143 (39). The p ecise in ol emen o 2-Cys P x o e oxida ion in plan hyd ogen pe oxide signaling emains o be es ablished. No a- bly, hyd ogen pe oxide o igina ing speci ically om chlo o- plas s has been ecognized o play a c i ical ole in he esponse and adap a ion o plan s o ele a ed ligh in ensi ies (40, 41). Mo eo e , an S x-de icien mu an o A abidopsis shows inc eased exp ession o he APX2 gene (42), hus sugges ing ha he o e oxida ion s a us o chlo oplas 2-Cys P x is in ol ed in hyd ogen pe oxide signaling in plan s. In mamma- lian cells, inac i a ion o P x I allows hyd ogen pe oxide accu- mula ion and cell signaling; howe e , inac i a ion occu s by phospho yla ion, no by o e oxida ion (43). In his ega d, he au ophospho yla ion o 2-Cys P x om apeseed is wo h men- ioning, al hough phospho yla ion occu s on sul inic and sul- onic o ms o he esol ing cys eine esidue (44). The e o e, mo e s udies a e equi ed o es ablish he ela ionship be ween edox s a us and phospho yla ion and he e ec o hese mod- i ica ions on hyd ogen pe oxide-dependen signaling in plan s. Thesimila i ieso he chlo oplas and Anabaena an ioxidan sys ems, based on sensi i e 2-Cys P x and he absence o ca a- lase ac i i y, sugges ha plan s ha e adop ed a low e iciency mechanism o pe oxide de oxi ica ion. In con as , his mech- anism, which ensu es apid inac i a ion and eac i a ion o he pe oxidase, would allow he ansien inc ease o hyd ogen pe - oxide le els and hus i s use as second messenge , in line wi h he impo an signaling ac i i y o his molecule in euka yo es. Acknowledgmen —We hank Rocio Rod iguez o excellen echnical assis ance. 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