Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b 559 from higher plants

1 Recons i u ion, spec oscopy and edox p ope ies o he pho osyn he ic ecombinan cy och ome b559 om highe plan s. Ma ía A. Lujána, Jesús I. Ma ínezb, Pablo J. Alonsob, Fe nando Gue e oc, Me cedes Roncelc, José M. O egac, Inmaculada Y uelaa and Ra ael Pico ela*. aEs ación Expe imen al de Aula Dei (EEAD), Consejo Supe io de In es igaciones Cien í icas (CSIC), Ca e e a Mon añana 1005, E-50059 Za agoza, Spain. bIns i u o Ciencia de Ma e iales de A agón (CSIC-Uni e sidad de Za agoza), C/ Ped o Ce buna 12, E-50009 Za agoza, Spain. cIns i u o de Bioquímica Vege al y Fo osín esis (CSIC-Uni e sidad de Se illa), C/ Amé ico Vespucio 49, E- 41092 Se illa, Spain. *Co esponding au ho : Ra ael Pico el, Es ación Expe imen al de Aula Dei, Ca e e a Mon añana 1005, E-50059 Za agoza, Spain. Phone: 34-976-716053; FAX: 34-976-716145; e- mail:[email p o ec ed]. 2 Abs ac A s udy o he in i o econs i u ion o suga bee cy och ome b559 o he pho osys em II is desc ibed. Bo h α and β cy och ome subuni s we e i s cloned and exp essed in Esche ichia coli. In i o econs i u ion o his cy och ome was ca ied ou wi h pa ially pu i ied ecombinan subuni s om inclusion bodies. Recons i u ion wi h comme cial heme o bo h (αα) and (ββ) homodime s and (αβ) he e odime was possible, he la e being mo e e icien . The abso p ion spec a o hese econs i u ed samples we e simila o ha o he na i e he e odime cy och ome b559 o m. As shown by elec on pa amagne ic esonance and po en iome y, mos o he econs i u ed cy och ome co esponded o a low spin o m wi h a midpoin edox po en ial +36 mV, simila o ha om he na i e pu i ied cy och ome b559. Fu he mo e, du ing he exp ession o suga bee and Synechocys is sp. PCC 6803 cy och ome b559 subuni s, pa o he p o ein subuni s we e inco po a ed in o he hos bac e ial inne memb ane, bu only in he case o he β subuni om he cyanobac e ium he o ma ion o a cy och ome b559-like s uc u e wi h he bac e ial endogenous heme was obse ed. The eason o ha su p ising esul is unknown. This in i o o med (ββ) homodime cy och ome b559-like s uc u e showed simila abso p ion and elec on pa amagne ic esonance spec al p ope ies as he na i e pu i ied cy och ome b559. A highe midpoin edox po en ial (+126 mV) was de ec ed in he in i o o med p o ein compa ed o he in i o econs i u ed o m, mos likely due o a mo e hyd ophobic en i onmen imposed by he lipid memb ane su ounding he heme. Keywo ds: Cy och ome b559, elec on pa amagne ic esonance, econs i u ion, edox i a ion. Abb e ia ions Abs Abso bance BCA bicinch oninic acid Cy cy och ome 3 β-DM n-dodecyl-β-D-mal oside DEAE die hyl aminoe hyl cellulose Eh ambien edox po encial Em midpoin edox po en ial EPR elec on pa amagne ic esonance ε ex inc ion coe icien HEPES 4-(2-hyd oxye hyl)-1-pipe azinee hanesul onic acid HP high po encial HS high spin IP in e media e po encial IPTG isop opyl β-D-1- hiogalac opy anoside KDS po assium dodecyl sulpha e LHCP ligh -ha es ing chlo ophyll-p o ein LP low po en ial LS low spin MBP mal ose-binding p o ein MES 2-(N-Mo pholino)e hanesul onic acid OD op ical densi y PAGE polyac ylamide gel elec opho esis PMSF phenylme hanesul onyl luo ide PS pho osys em SDS sodium dodecyl sulpha e TRIS is(hyd oxyme hyl)aminome hane 4 In oduc ion Cy och ome b559 (Cy b559) is an in eg al componen o he pho osys em II (PSII) eac ion cen e (S ewa and B ud ig 1998). I comp ises wo small polypep ides, α (9 kDa) and β (4.5 kDa) subuni s, encoded by psbE and psbF genes, espec i ely. Each subuni con ains one alpha- helix ha spans he hylakoid memb ane, wi h he N- e minus in he s omal side (Gusko e al. 2009). The b- ype heme is coo dina ed o wo his idines in a plana axial s uc u e (Babcock e al. 1985; Ga cía-Rubio e al. 2003), one om each subuni , and i is loca ed owa ds he s omal side (Pico el e al. 1994; Gusko e al. 2009). Despi e many a emp s, Cy b559 unc ion emains unclea . I has been demons a ed ha Cy b559 is essen ial o he co ec assembly o he PSII (Pak asi e al. 1989; Swia ek e al. 2003), al hough i is no in ol ed in he p ima y elec on ans e wi hin PSII (S ewa and B ud ig 1998). One o he mos accep ed hypo heses pu o wa d sugges s i s in ol emen in PSII p o ec ion agains pho oinhibi ion (S ewa and B ud ig 1998; Hung e al. 2010). Cy och ome b559 has singula edox p ope ies among b- ype cy och omes. I exhibi s se e al midpoin edox po en ial (Em) o ms (C ame and Whi ma sh 1977; O ega e al. 1988; Thompson e al. 1989; Roncel e al. 2001): a high-po en ial o m (HP, Em ≈ +400 mV), an in e media e-po en ial o m (IP, Em ≈ +200-150 mV), and a low-po en ial o m (LP, Em ≈ +100 mV). The HP o m is e y labile as i has only been obse ed in in ac chlo oplas s and some isola ed PSII p epa a ions (S ewa and B ud ig 1998). Pu i ied Cy b559 displayed only he LP o m (Me z e al. 1983). The molecula mechanisms esponsible o hese singula edox p ope ies a e mainly unknown, al hough se e al hypo heses ha e been p oposed (Me z e al. 1983; Babcock e al. 1985; Roncel e al. 2001; Kaminskaya e al. 2007; Shibamo o e al. 2008). The deg ee o he heme exposu e o sol en s and i s en i onmen al hyd ophobici y seem o modula e he Cy b559 edox p ope ies (O ega e al. 1988; Ahmad e al. 1993; Kaminskaya e al. 1999; Roncel e al. 2001). 5 Cy och ome b559 is a pa amagne ic species, hus elec on pa amagne ic esonance (EPR) spec oscopy is a sui able echnique o cha ac e ize his me allop o ein. The p incipal alues o he g- enso a e gZ ≈ 3.05-2.9, gY ≈2.26-2.15, and gX ≈ 1.5-1.4, which co espond o a low spin (LS) heme cen e (Babcock e al. 1985; S ewa and B ud ig 1998; Y uela e al. 2003). Some in i o and in i o s udies ha e been ca ied ou wi h he β subuni alone. I was ound ha he β subuni was able o unde go (ββ) homodime iza ion and bound he heme g oup o make a Cy b559-like s uc u e. I showed spec al and edox p ope ies simila o hose o na i e Cy b559. In i o econs i u ions wi h comme cial heme and he β subuni om he cyanobac e ium Synechocys is sp. PCC 6803 we e desc ibed, ei he om chemically syn hesized pep ide (F ancke e al. 1999) o om he inclusion bodies o Esche ichia (E.) coli exp essing β subuni in (P odohl e al. 2005). Fu he mo e, in i o o ma ion o he (ββ) homodime ic Cy b559-like s uc u e was epo ed when he β subuni om Synechocys is (P odohl e al. 2005) o Synechococcus sp. PCC 7002 (Yu e al. 2003) was exp essed in E. coli. The polypep ide was in eg a ed in o he hos bac e ial inne memb ane as a (ββ) homodime , and hen i bound bac e ial endogenous heme o make a Cy b559-like s uc u e. In he p esen s udy, we i s epo he exp ession in E. coli o he wo Cy b559 subuni s α and β om plan species, and he psbE gene om a cyanobac e ium. Wi h he ecombinan subuni s om suga bee (MBP-Rsubα and MBP-Rsubβ), we s udied he abili y o in i o econs i u ion o αα, ββ and αβ o ms, he he e odime being mo e e icien . We also desc ibe he inse ion o α and β subuni s om suga bee and Synechocys is (MBP-Ssubα and MBP- Ssubβ), and he β subuni om maize (MBP-Msubβ) in E. coli inne cy oplasmic memb ane du ing hei exp ession. Only he β subuni om he cyanobac e ium was able o bind heme o o m a Cy b559-like s uc u e in i o. The spec oscopic and edox p ope ies o he ob ained o ms a e desc ibed. 6 Ma e ials and me hods Cloning and exp ession o he cy och ome b559 subuni s. The psbE and psbF genes om suga bee and Synechocys is and psbF gene om maize we e cloned and exp essed in E. coli as usion p o eins using s anda d p ocedu es. Genes we e ob ained om genomic DNA by PCR wi h speci ic p ime s, con aining a BamHI si e o wa d oligonucleo ides and a HindIII si e e e se p ime s. Each gene was cloned in o pMAL-c2X exp ession ec o and he sequence o each inal cons uc was con i med by DNA sequencing. These cons uc ions will gi e usion p o eins composed o MBP (mal ose-binding p o ein), a p o ein ca ie , and α o β Cy b559 subuni s. Esche ichia coli TB1 compe en cells we e ans o med wi h he cons uc s o exp ession o each usion p o ein. A p e-cul u e was g own o e nigh om a selec ed single colony a 37 ºC in LB b o h (Mille ) medium in he p esence o 100 µg ml-1 ampicillin. A la ge olume o LB b o h wi h ampicillin was inocula ed wi h he p e-cul u e on he nex day and i was g own a 37 ºC un il he OD600nm eached 0.6 uni s. Then 0.5 mM isop opyl β-D-1- hiogalac opy anoside (IPTG) was added o he cul u e o induce he exp ession o he usion p o eins o 3 h a 37 ºC, wi h he excep ion o he MBP-Ssubα ha was exp essed wi h 1 mM IPTG o 17 h a 18 ºC. A e induc ion, cells we e ha es ed by cen i uga ion a 10,000g o 5 min and ozen a -20 ºC un il use. Fo p o ein pu i ica ion, he bac e ial pelle was esuspended in 50 mM HEPES, pH 7.5, 10 mM EDTA, and b oken by sonica ion (Ul asonic P ocesso XL 2020 Misonix, Fa mingale, NY USA) o 45 min (90 s pulses wi h 60 s in e als) wi h sample ecipien on ice o a oid hea ing. Cell ex ac s we e cla i ied by cen i uga ion a 15,000g o 10 min a 4 ºC, ob aining he insoluble ma e ial wi h he inclusion bodies as a pelle . This pelle was sa ed and he supe na an cen i uged again a 40,000g o 45 min a 4 ºC o sedimen small cy oplasmic memb ane agmen s, which we e esuspended in he same bu e and analyzed by UV-Vis abso p ion and EPR spec oscopies, and edox po en iome ic i a ion. 7 The sa ed pelle om he i s cen i uga ion con aining he inclusion bodies was washed once by cen i uga ion (10,000g o 5 min a 4 ºC) wi h 50 mM HEPES, pH 7.5, 10 mM EDTA, 1% (w/ ) T i on X-100, esuspended wi h 20 mM TRIS-HCl, pH 8.0, 50 mM sodium dodecyl sulpha e (SDS) and sa ed o make subsequen ly in i o econs i u ions. To al p o ein concen a ion was de e mined using he BCA eagen (Pie ce The mo Scien i ic, Rock o d, IL USA). P o eins we e sepa a ed on SDS-PAGE [12% o 20% (w/ ) ac ylamide and 4 M u ea] and e ealed by Coomassie B illian Blue s aining. In i o econs i u ion In i o he e odime ic Cy b559 econs i u ion was done using equimola amoun s o MBP-Rsubα and MBP-Rsubβ usion p o eins ob ained as explained abo e. The de e gen SDS o he usion p o ein mix u e was exchanged wi h 0.15 mM n-dodecyl β-D-mal oside (β-DM) by p ecipi a ing wi h 50 mM KCl. A e incuba ing o 10 min on ice, he p ecipi a ed po assium dodecyl sulpha e (KDS) was emo ed by cen i uga ion a 6,000g o 15 min a 4 ºC. Finally, equimola amoun s o heme om chemical hemin chlo ide (Fluka, Buchs, Swi ze land) dissol ed as desc ibed by K oliczewski and Szczepaniak (2002), we e added o he usion p o ein mix u e. A e in i o econs i u ion, p o ease clea age was pe o med wi h Fac o Xa o elease he Cy b559 subuni s om he MBP. Clea age was done in 20 mM TRIS-HCl, pH 8.0, 1 mM CaCl2, 0.15 mM β-DM wi h 10 uni s o Fac o Xa pe mg o usion p o ein a 22 ºC o 4 h. The eac ion was s opped by adding 1 mM PMSF p o ease inhibi o . The esul an clea age mix u e was pu i ied by weak anionic-exchange ch oma og aphy wi h a TSK Toyopea l DEAE 650s (TOSOH Bioscience GmbH, S u ga , Ge many) column p e-equilib a ed wi h 20 mM TRIS-HCl, pH 8.0, and 0.15 mM β-DM. A e sample loading, he column was washed wi h i e column olumes o he same bu e , and he ma e ial elu ed wi h a 0-500 mM NaCl con inuous g adien in he same bu e a a low a e o 0.5 ml min-1 in 1-ml ac ions. F ac ions we e analyzed by SDS-PAGE and Coomassie B illian Blue s aining, and hose con aining Cy b559 subuni s we e pooled and 8 concen a ed using a Cen ip ep o Cen icon 3000 NMWL il e (Millipo e, Bille ica, MA USA). Since some hemes could ha e been de ached du ing he ch oma og aphy, comme cial heme was added again in excess o he concen a ed sample o ensu e maximum econs i u ion, and, immedia ely a e , he mix u e was passed h ough h ee consecu i e desal ing PD-10 columns (GE Heal hca e) o emo e mos o ee heme (K oliczewski and Szczepaniak 2002). The inal sample was concen a ed en imes using a Cen icon 3000 NMWL il e ube, and he deg ee o Cy b559 econs i u ion was de e mined by UV-Vis spec oscopy. In i o (αα) and (ββ) homodime ic Cy b559 econs i u ions we e ob ained in he same way bu using one ype o usion p o ein only. Abso p ion spec oscopy Visible abso p ion spec a in he 400-600 nm ange we e ob ained wi h a Beckman DU 640 spec opho ome e (Beckman Coul e , B ea, CA USA). Samples we e measu ed ei he in ai - oxidized o educed wi h 10 mM sodium di hioni e. The spec a we e compa ed a he α band a a ound 559 nm o he educed minus oxidized di e en ial abso p ion spec a. Two ex inc ion coe icien s (ε) we e used o de e mine he ex en o he cy och ome econs i u ion using he di e ence abso p ion spec a, i.e., ε559.5nm - 577nm = 21.5 mM-1 cm-1 o ε559.5nm - isosbes ic poin = 17.5 mM-1 cm-1 (S ewa and B ud ig 1998). Elec on pa amagne ic esonance EPR measu emen s we e eco ded wi h a B uke ESP380E spec ome e (B uke , Ka ls uhe, Ge many) wo king a he X-band. Fo low- empe a u e measu emen s, an Ox o d CF935 liquid helium con inuous- low c yos a (Ox o d Ins umen s, Eynsham, UK) was used. Typical condi ions o con inuous-wa e EPR (CW-EPR) measu emen s we e: empe a u e, 15K; mic owa e equency, 9.70 GHz; mic owa e powe , 3.2 x 10-2 mW; modula ion ampli ude, 3.0 Gauss. Two-pulse echo induced EPR (2p ei-EPR) expe imen s we e pe o med by using he 9 (п/2 - т - п - т) sequence, and de ec ing he echo in ensi y as a unc ion o he applied magne ic ield. The expe imen al condi ions we e 6K and 9.78 GHz. Some ei-EPR spec a showed a spu ious con ibu ion mos p obably om a Cu(II) species (see Resul s). In o de o make he con ibu ions om heme cen e s easie o see, a nume ical sub ac ion o his spu ious signal was pe o med. F om a sample whe e he ela i e in ensi y o he heme signals was low, he Cu(II) signal in he ield in e al (270-360 mT) was isola ed. This signal was sub ac ed om he measu ed spec a o he Cy b 559 econs i u ed samples. Addi ionally, a nume ical “adjacen a e aging” il e was used in o de o imp o e he signal- o- noise a io wi hou losing he signals esolu ion. Po en iome ic edox i a ions Po en iome ic edox i a ions we e ca ied ou basically as desc ibed by Gue e o e al. (2011). Fo i a ions, samples we e suspended in 2.5 ml bu e con aining 40 mM MES-NaOH, pH 6.5, 0.587 mM β-DM, and he ollowing edox media o s: 10 µM 2,5-dime hyl-p-benzoquinone (E’m7 = +180 mV), 20 µM o-naph oquinone (E’m7 = +145 mV), 2.5 µM N-me hyl-phenazonium me hosul a e (E’m7 = +80 mV) and 20 µM du oquinone (E’m7 = +5 mV). Expe imen s we e done a 20 ºC unde a gon a mosphe e and con inuous s i ing. Reduc i e i a ions we e pe o med by i s oxidizing wi h 25 µM po assium e icyanide and hen educing i s epwise wi h small aliquo s o 0.1 M sodium di hioni e. A e addi ion o sodium di hioni e, he abso p ion spec um be ween 500-600 nm ange and he edox po en ial o he solu ion we e simul aneously eco ded by using, espec i ely, a SLM Aminco DW2000 UV-Vis spec opho ome e and a Me ohm po en iome e (Me ohm L d., He isau, Swi ze land) p o ided wi h a combined P - Ag/AgCl mic oelec ode (Mic oelec odes Inc, Bed o d, NH USA) p e iously calib a ed agains a sa u a ed solu ion o quinhyd one (E’m7 = +280 mV a 20 ºC). Di e en ial spec a o Cy b559 we e ob ained by sub ac ing he absolu e spec a eco ded a each Eh du ing i a ions om he spec a o he ully oxidized cy och ome. The abso bance di e ence a 559 nm minus 570 nm 16 al eady ha e he Phe codon a ha posi ion). This change due o RNA-edi ing ha occu s in ce ain highe plan s is no possible in bac e ia (Maie e al. 1996). Thus β subuni om suga bee exp essed in bac e ia main ained a Se a posi ion 26 ins ead o a Phe. To de e mine i he p esence o Se 26 in he ecombinan β subuni sequence impai ed i s econs i u ion, we exp essed he β subuni om maize (MBP-Msubβ) in E. coli (no e ha psbF gene om maize al eady encodes Phe esidue a posi ion 26). When he edox di e en ial abso p ion spec um o memb ane agmen s con aining he MBP-Msubβ om maize was compa ed wi h ha o he β subuni om suga bee , no di e ences we e obse ed be ween bo h samples (da a no shown). Thus he Phe26 esidue was no essen ial o in i o o ma ion wi h he β subuni . Since β subuni om Synechocys is was he only polypep ide able o o m spon aneously a Cy b559-like s uc u e in he hos E. coli inne memb ane, we u he cha ac e ized such a p o ein s uc u e by EPR spec oscopy and po en iome y. To dis inguish be ween he p ope ies o he o med (ββ) Cy b559-like s uc u e om o he b- ype endogenous cy och omes and o he po en ial in e e ences induced by he exp essed p o ein ca ie , MBP, we also s udied he bac e ial memb ane agmen s exp essing he emp y pMAL-c2X ec o (nega i e con ol). CW-EPR spec a (Fig. 7a) showed ea u es a ge = 6.0, ge = 4.3 and ge = 2.0 alues ha we e p esen bo h in he nega i e con ol and in he memb ane agmen s con aining (ββ) homodime . They co esponded o HS heme, non-heminic i on o endogenous adicals loca ed in he E. coli inne memb ane. Besides, LS heme con ibu ions we e also de ec ed bo h in he nega i e con ol and in he memb ane agmen s con aining (ββ) homodine . A pa o hese signal in ensi ies come om endogenous heme cen e s loca ed in he E. coli inne memb ane bu he LS heme con ibu ion in he CW-EPR spec um o he nega i e con ol (no shown) was clea ly much less in ense han he one de ec ed o memb ane agmen s con aining (ββ) homodime s. When he CW-spec um o he nega i e con ol was sub ac ed om ha o he 17 (ββ) homodime sample (Fig. 7a), he emaining ea u es a ge = 2.96 and ge = 2.28 should co espond o he (ββ) homodime ic Cy b559–like s uc u e (Fig. 7a). The ei-EPR spec um showed again in ense ea u es a ge ≈ 2.08 (Cu(II) signal) and ge = 2.0 (signal om endogenous ee adicals). These ea u es a e unca ed in Fig. 7b o a shake o cla i y. Besides, he ei-EPR spec um displayed cha ac e is ic ea u es (ge = 2.96, ge = 2.28, and ge = 1.5), ypical o a LS heme analogous o ha p e iously epo ed o na i e Cy b559 (Babcock e al. 1985; S ewa and B ud ig 1998; Y uela e al. 2003). Thus his sample showed only a LS o m due o (ββ) homodime ic Cy b559-like s uc u e wi hin E. coli memb ane ha co esponds o a heme wi h a plana axial bis-his idine coo dina ion as in he case o na i e Cy b559 (Babcock e al. 1985; S ewa and B ud ig 1998; Y uela e al. 2003). Po en iome ic educ i e i a ions we e ca ied ou a pH 6.5 (Fig. 8) in he same edox condi ions han hose o in i o econs i u ion. E. coli inne memb ane agmen s con aining β subuni exhibi ed wo edox species wi h di e en Em (i.e., +58 mV and +126 mV) (Fig. 8). No e ha he measu emen s we e achie ed in E. coli memb ane agmen s, whe e endogenous cy och omes we e also p esen as men ioned abo e. As in he case o abso p ion and EPR measu emen s, hese endogenous hemop o eins should also be de ec ed in he po en iome ic measu emen s. As a con ol, po en iome ic educ i e i a ions o E. coli inne memb ane agmen s con aining MBP only we e ca ied ou , and only one Em o +60 mV was calcula ed (Fig. 8). These esul s sugges ed ha Em = +58 mV o m, ound in memb ane agmen s con aining β subuni was due o bac e ial endogenous cy och omes, and he Em = +126 mV o m co esponded o he o ma ion o (ββ) homodime ic Cy b559-like s uc u e. The highe Em (+126 mV) ob ained in he bac e ial memb anes should no be due o he ac ha he β polypep ide is o ming a usion p o ein wi h he MBP, a soluble moie y. P elimina y expe imen s indica ed ha he usion p o eins a e ancho ed wi hin he bac e ial cy oplasmic memb ane by he alpha-helix o he Cy b559 subuni s since p o ease ea men ac ually libe a ed he MBP 18 moie y, emaining he alpha-helix in he memb ane pelle a e cen i uga ion (da a no shown). So he soluble moie y o he usion p o eins is e y well sepa a ed om he cy och ome subuni s making i di icul o ha e any signi ican in luence on he edox p ope ies o in i o o med Cy b559-like s uc u e. Discussion In ecen yea s, in i o econs i u ions o a ious b- ype cy och omes ha e been epo ed. Fo ins ance, ecombinan memb ane p o eins as Cy b5 (Mul ooney and Waskell 2000), Cy b6 (K oliczewski and Szczepaniak 2002) and (ββ) homodime Cy b559–like s uc u e (P odohl e al. 2005), ha e been desc ibed. In he p esen s udy, we epo o he i s ime in i o econs i u ion o he pho osyn he ic Cy b559, a cy och ome comp ised o wo di e en p o ein subuni s ha coo dina e a heme g oup. I has o be no ed ha he spec al α-band a ound 559 nm o he he e odime appea ed somewha less dis o ed and wi h highe ampli ude han hose om he homodime s, indica ing ha (αβ) he e odime iza ion domina es o e (αα) o (ββ) homodime iza ion. This may explain why na u e has selec ed he he e odime as he unc ional o m in oxygenic pho osyn hesis. Howe e , we s ill canno disca d he o ma ion o some (αα) and (ββ) homodime s du ing he in i o (αβ) he e odime econs i u ion. We ha e ied o sepa a e he di e en po en ial dime ic o ms (αβ, αα, ββ) o he econs i u ion mix u e by using na i e PAGE gel wi h mild condi ions and p obe wi h he co esponding an ibodies, bu all dime ic o ms we e des oyed du ing he elec opho esis, he subuni s α and β appea ing as sepa a ed bands. Such ins abili y o he in i o econs i u ed Cy b559 is no o ally su p ising, conside ing he 3-D s uc u e o his cy och ome, whe e he h ee moie ies (α, β, heme) a e highly exposed o he elec opho esis eac an s. Besides he na i e EPR o m LS1, wo addi ional o ms, HS and LS2 we e ound in he econs i u ed he e odime ic sample om plan s (Fig. 4). Bo h HS and LS2 o ms ha e also 19 been ound in na i e Cy b559 (Loew 1983; Scheid and Gou e man 1983; Shu alo e al. 1995; K opache a e al. 2003) and, acco ding o he ea u es obse ed in o he me allop o eins such as ca alase, haemoglobin and myoglobin (Blumbe g and Peisach 1971; Fiege e al. 1995), assigned o heme dis o ed en i onmen (HS) and o heme cen e s wi h one o he coo dina ed his idines displaced by hyd oxyl g oup (LS2). The in ensi y o he LS2 signal in he na i e Cy b559 EPR spec a depended on sample in eg i y and ea men s (Fiege e al. 1995; Bianche i e al. 1998). A model has been sugges ed in which he wo subuni s emained linked, bu one his idine ligand was eplaced by an –OH g oup as second axial ligand (Shu alo e al. 1994). I is well possible ha some he e odime s a e no pe ec ly assembled in i o, because he p o ein subuni s a e ee in solu ion, in con as o mo e es ic ed condi ions wi hin he biological memb ane. The de e gen p esen in he solu ion could also add some s uc u al dis o ion, al hough small amoun s o SDS de e gen was p o ed e en o acili a e he in i o econs i u ion (Webe e al. 2011). The Em (+36 mV) o ou in i o econs i u ed samples was e y simila o ha ound in isola ed na i e Cy b559 (Ma suda and Bu le 1983; O ega and He ás 1989), hough his edox po en ial is in luenced by he pH and he hyd ophobici y su ounding he Cy b559 heme cen e (O ega e al. 1988; Ahmad e al. 1993; Kaminskaya e al. 1999; Roncel e al. 2001; Webe e al. 2011). Exp essed o eign in eg al memb ane p o eins in E. coli ha e a na u al endency o agg ega e in o inclusion bodies o inse in o he bac e ial inne memb ane (D ew e al. 2003). Examples o spon aneous inse ion in o he bac e ial memb ane a e he ecombinan LHCP (Koho n and Auchincloss 1991), Cy (Ro hs ein e al. 1985), Cy b5 (Smi h e al. 1994) and Cy b6 (K oliczewski e al. 2005). Bu hese h ee cy och omes we e made o a single polypep ide, and we e ob ained as holop o ein using bac e ial endogenous heme. Recen wo ks ha e also desc ibed ans o man bac e ial memb anes wi h he Cy b559 β subuni om Synechocys is 20 (P odohl e al. 2005) and Synechococcus (Yu e al. 2003), wi h spec al p ope ies analogous o hose o na i e Cy b559. Acco ding o ou SDS-PAGE analysis, bo h Cy b559 subuni s om he di e en o ganisms used in he p esen s udy we e inse ed spon aneously in o he bac e ial inne memb ane (Fig. 1). Howe e , among all Cy b559 subuni s analyzed in he p esen s udy, β subuni om Synechocys is was he only polypep ide able o o m a Cy b559-like s uc u e spon aneously in E. coli inne memb ane. The nega i e esul s ob ained wi h α subuni om all o ganisms es ed, sugges ha he e a e some cons ains o he co ec heme assembly independen ly o p o ein inse ion wi hin he bac e ial memb ane. This nega i e esul was no due o less s abili y o he ecombinan α polypep ide wi hin he bac e ial memb ane due o, o ins ance, speci ic memb ane p o ease deg ada ion since i s size emained he same as he na i e one (Fig. 1). No e ha we also de ec ed a poo e in i o econs i u ion capaci y o his subuni (see Fig. 3). A p esen we do no ha e any clue o explain why o e exp essed β subuni om plan suga bee unlike Synechocys is was unable o o m a Cy b559-like s uc u e in E. coli inne memb ane, hough i s sequence is highly conse ed and in bo h cases he subuni s we e in eg a ed in he bac e ial memb ane as ull polypep ides. The chlo oplas ic o igin o ou plan p o ein subuni s does no seem o accoun o i since ecombinan Cy b6 om spinach was success ully econs i u ed in bac e ia (K oliczewski e al. 2005). EPR analysis o bac e ial inne memb ane agmen s con aining he β subuni om Synechocys is showed only a LS1 o m, wi h simila spec oscopic ea u es o hose o na i e Cy b559 (Babcock e al. 1985; S ewa and B ud ig 1998; Y uela e al. 2003). This p o es he heme coo dina ion o in i o o med (ββ) homodime . I is wo h o no ing ha LS2 was no de ec ed in hese samples, indica ing ha he o ma ion o a dis o ed Cy b559 was es ic ed wi hin he biological memb ane mos p obably due o less hyd oxyla ion ac i i y wi hin he hyd ophobic memb ane ma ix. In addi ion, he EPR ea u es o his LS1 o m shows a small 21 shi ing when compa ed o hose o he LS1 o m ound in in i o econs i u ed Cy b559. These disc epancies may be connec ed wi h some small di e ences in he en i onmen su ounding he LS heme cen e s in bo h cases (Y uela e al. 2003). Fu he mo e, p e ious Em measu ed o he (ββ) homodime (+50 mV) we e smalle han ha ob ained in he p esen wo k (+126 mV) (F ancke e al. 1999; Yu e al. 2003). I has o be no ed ha hese po en iome ic measu emen s we e ca ied ou in samples whe e he (ββ) homodime was ou side o a biological memb ane and, he e o e, close o ha o ou in i o econs i u ion. The obse ed di e ences indica e he impo an ole o he en i onmen su ounding he heme on i s Em alue (O ega e al. 1988; Ahmad e al. 1993; Kaminskaya e al. 1999; Roncel e al. 2001). In ac , he heme en i onmen o he in i o o ma ion and he na u al hylakoid memb ane condi ions is mo e hyd ophobic han he aqueous bu e solu ions used o in i o econs i u ion. In e es ingly, he na i e Cy b559 LP o m in PSII memb anes exhibi s a edox po en ial alue o +110 mV (Roncel e al. 2001), simila o ha measu ed in his wo k in bac e ial inne memb ane agmen s. Concluding, we ha e de eloped a me hodology o econs i u e he αβ he e odime ic o m o a plan Cy b559. This can be use ul ool o u u e s udies o elucida e he mechanism by which Cy b559 ma u es, and o explo e he molecula mechanisms ha egula e he in iguing edox po en ial a iabili y o his elusi e me allop o ein. This me hodology can e en be mo e powe ul i used oge he wi h si e-di ec ed mu agenesis echniques, and he applica ion o ad anced spec oscopic echniques wi h no in e e ences om o he e apy ol molecules such as chlo ophylls as no mally occu ed when using pho osyn he ic ma e ials. Fu u e wo k will also aim o de e mine he di e en beha iou o α subuni compa ed o β subuni o in i o o ma ion despi e hei s uc u al simila i y. Acknowledgemen s 22 This wo k was suppo ed by G an s AGL2008-00377, MAT2008-03461, and BFU2007-68107- C02-01 om he Spanish Minis y o Science and Inno a ion (MICINN), PADI CVI-261 om he Andalusia Regional Go e nmen , and DGA-GC E33 and DGA-GE B18 om A agon Regional Go e nmen . All hese G an s we e pa ially inanced by he EU FEDER P og am. M. A. 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