Is the Redox State of the ci Heme of the Cytochrome b6f Complex Dependent on the Occupation and Structure of the Qi Site and Vice Versa?
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.016709
ISSN1083-351X
AutoresAgnès de Lacroix de Lavalette, Lise Barucq, Jean Alric, Fabrice Rappaport, Francesca Zito,
Tópico(s)Porphyrin and Phthalocyanine Chemistry
ResumoOxidoreductases of the cytochrome bc1/b6f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b6f has revealed the presence in the Qi site of an atypical c-type heme, heme ci. Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the ci heme and its interaction with the Qi site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe40 from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the ci heme midpoint potential (from +100 mV to −200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b6f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis. Oxidoreductases of the cytochrome bc1/b6f family transfer electrons from a liposoluble quinol to a soluble acceptor protein and contribute to the formation of a transmembrane electrochemical potential. The crystal structure of cyt b6f has revealed the presence in the Qi site of an atypical c-type heme, heme ci. Surprisingly, the protein does not provide any axial ligand to the iron of this heme, and its surrounding structure suggests it can be accessed by exogenous ligand. In this work we describe a mutagenesis approach aimed at characterizing the ci heme and its interaction with the Qi site environment. We engineered a mutant of Chlamydomonas reinhardtii in which Phe40 from subunit IV was substituted by a tyrosine. This results in a dramatic slowing down of the reoxidation of the b hemes under single flash excitation, suggesting hindered accessibility of the heme to its quinone substrate. This modified accessibility likely originates from the ligation of the heme iron by the phenol(ate) side chain introduced by the mutation. Indeed, it also results in a marked downshift of the ci heme midpoint potential (from +100 mV to −200 mV at pH 7). Yet the overall turnover rate of the mutant cytochrome b6f complex under continuous illumination was found similar to the wild type one, both in vitro and in vivo. We propose that, in the mutant, a change in the ligation state of the heme upon its reduction could act as a redox switch that would control the accessibility of the substrate to the heme and trigger the catalysis. The cytochrome b6f complex of oxygenic photosynthesis is the integral membrane protein, the quinol:plastocyanin oxido-reductase activity of which allows the linear electron flow between the two photosystems (PSI and PSII). 4The abbreviations used are: PSIphotosystem IPSIIphotosystem IIbHhigh potential heme bbLlow potential heme bNQNO2-n-nonyl-4-hydroxyquinoline N-oxidePQplastoquinonecytcytochromeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineWTwild type. 4The abbreviations used are: PSIphotosystem IPSIIphotosystem IIbHhigh potential heme bbLlow potential heme bNQNO2-n-nonyl-4-hydroxyquinoline N-oxidePQplastoquinonecytcytochromeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineWTwild type. The turnover of the cytochrome b6f complex depends on the steady state of its redox partners, the liposoluble plastoquinol (PQH2 reduced and protonated plastoquinone PQ) formed by the PSII, and the hydrosoluble plastocyanin oxidized by the PSI. In the Qo site, exposed to the lumenal side, the quinol is oxidized, and this oxidation is coupled to the release of two protons into the lumen. The two electrons provided by the quinol are transferred along two bifurcated pathways, the high and low potential chains. The high potential chain involves two lumenal redox partners, the membrane-anchored flexible Rieske [2Fe-2S] cluster and the cytochrome f, which ultimately interacts with the soluble plastocyanin. In the low potential chain, electrons are transferred to the stroma via the low and high potential b hemes (bL and bH) of the transmembrane b6 subunit. Two turnovers of the cyt b6f complex lead to the reduction of the low potential chain, thereby allowing the reduction of a quinone molecule in the stromal Qi pocket. This mechanism, which recycles reducing equivalents, is referred to as the "Q cycle," initially described by Mitchell (1Mitchell P. FEBS Lett. 1975; 56: 1-6Crossref PubMed Scopus (316) Google Scholar) and modified later by Crofts et al. and others (2Crofts A.R. Meinhardt S.W. Biochem. Soc. Trans. 1982; 10: 201-203Crossref PubMed Scopus (39) Google Scholar, 3Osyczka A. Moser C.C. Dutton P.L. Trends Biochem. Sci. 2005; 30: 176-182Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). photosystem I photosystem II high potential heme b low potential heme b 2-n-nonyl-4-hydroxyquinoline N-oxide plastoquinone cytochrome N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine wild type. photosystem I photosystem II high potential heme b low potential heme b 2-n-nonyl-4-hydroxyquinoline N-oxide plastoquinone cytochrome N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine wild type. Although this quinol:cytochrome oxidoreductase activity is involved in both the respiratory and photosynthetic electron transfer chains, recent x-ray data (4Kurisu G. Zhang H. Smith J.L. Cramer W.A. Science. 2003; 302: 1009-1014Crossref PubMed Scopus (596) Google Scholar, 5Stroebel D. Choquet Y. Popot J.L. Picot D. Nature. 2003; 426: 413-418Crossref PubMed Scopus (507) Google Scholar, 6Baniulis D. Yamashita E. Whitelegge J.P. Zatsman A.I. Hendrich M.P. Hasan S.S. Ryan C.M. Cramer W.A. J. Biol. Chem. 2009; 284: 9861-9869Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) have evidenced major structural differences between the b6f complex and its mitochondrial counterpart the bc1 complex (for reviews see Refs. 7Berry E.A. Guergova-Kuras M. Huang L.S. Crofts A.R. Annu. Rev. Biochem. 2000; 69: 1005-1075Crossref PubMed Scopus (397) Google Scholar, 8Cramer W.A. Yan J. Zhang H. Kurisu G. Smith J.L. Photosynth. Res. 2005; 85: 133-143Crossref PubMed Scopus (30) Google Scholar, 9Darrouzet E. Cooley J.W. Daldal F. Photosynth. Res. 2004; 79: 25-44Crossref PubMed Scopus (39) Google Scholar, 10Crofts A.R. Photosynth. Res. 2004; 80: 223-243Crossref PubMed Scopus (66) Google Scholar). Indeed an additional heme localized in close contact with heme bH stands as another putative electron carrier as proposed earlier by Lavergne (11Lavergne J. Biochim. Biophys. Acta. 1983; 725: 25-33Crossref Scopus (44) Google Scholar). Since it was brought to light by the x-ray studies, knowledge of the basic properties of this heme, named ci in reference to the Qi site (5Stroebel D. Choquet Y. Popot J.L. Picot D. Nature. 2003; 426: 413-418Crossref PubMed Scopus (507) Google Scholar) or cn in reference to its proximity to the negatively charged side of the membrane (4Kurisu G. Zhang H. Smith J.L. Cramer W.A. Science. 2003; 302: 1009-1014Crossref PubMed Scopus (596) Google Scholar), has significantly improved. The proteins involved in the assembly machinery of the heme have been identified in Chlamydomonas reinhardtii and Arabidopsis thaliana (12Kuras R. Saint-Marcoux D. Wollman F.A. de Vitry C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 9906-9910Crossref PubMed Scopus (60) Google Scholar, 13Lezhneva L. Kuras R. Ephritikhine G. de Vitry C. J. Biol. Chem. 2008; 283: 24608-24616Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Consistent with the structure, according to which the only axial ligand could be a water molecule interacting with the proponiate chain of the bH heme, the spectroscopic properties of this heme are those of a high spin heme (14Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15860-15865Crossref PubMed Scopus (72) Google Scholar, 15Baymann F. Giusti F. Picot D. Nitschke W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 519-524Crossref PubMed Scopus (36) Google Scholar). Evidences for a high spin heme covalently bound to the cytochrome b subunit were also found in Heliobacterium modesticaldum and Heliobacillus mobilis (16Ducluzeau A.L. Chenu E. Capowiez L. Baymann F. Biochim. Biophys. Acta. 2008; 1777: 1140-1146Crossref PubMed Scopus (19) Google Scholar). In the b6f complex from the oxygenic photosynthetic chain, EPR (15Baymann F. Giusti F. Picot D. Nitschke W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 519-524Crossref PubMed Scopus (36) Google Scholar) and structural data (17Yamashita E. Zhang H. Cramer W.A. J. Mol. Biol. 2007; 370: 39-52Crossref PubMed Scopus (96) Google Scholar) have shown that NQNO (2-n-nony l-4-hydroxyquinoline N-oxide), an inhibitor of the Qi pocket (18Musser S.M. Stowell M.H. Lee H.K. Rumbley J.N. Chan S.I. Biochemistry. 1997; 36: 894-902Crossref PubMed Scopus (44) Google Scholar, 19Zhang H. Primak A. Cape J. Bowman M.K. Kramer D.M. Cramer W.A. Biochemistry. 2004; 43: 16329-16336Crossref PubMed Scopus (37) Google Scholar), can act as an axial ligand to ci. This ligation is accompanied by a significant change in the redox properties of ci, because, in the presence of NQNO, at least two titrations waves were observed (13Lezhneva L. Kuras R. Ephritikhine G. de Vitry C. J. Biol. Chem. 2008; 283: 24608-24616Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 14Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15860-15865Crossref PubMed Scopus (72) Google Scholar), one with a midpoint potential (Em) similar to that observed in the absence of NQNO and the other with a midpoint potential downshifted by ∼250 mV. This, together with the widespread range of redox potential found for heme ci (11Lavergne J. Biochim. Biophys. Acta. 1983; 725: 25-33Crossref Scopus (44) Google Scholar, 14Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15860-15865Crossref PubMed Scopus (72) Google Scholar, 15Baymann F. Giusti F. Picot D. Nitschke W. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 519-524Crossref PubMed Scopus (36) Google Scholar, 20Joliot P. Joliot A. Biochim. Biophys. Acta. 1988; 933: 319-333Crossref Scopus (61) Google Scholar), points to a structural plasticity of the ci ligand network. This plasticity may arise from the unusual coordination properties of the heme ci. As a matter of fact, the x-ray models of the complex from C. reinhardtii and Mastigocladus laminosus evidenced a water or hydroxyl molecule as a fifth ligand. The sixth position of coordination is directed toward the Qi pocket and appears as free. Nevertheless, the side chain of Phe40 of subunit IV protrudes above the heme plane, leaving little space for any axial ligand to the heme ci. Besides, modeling a quinone analogue in the electron density found in the Qi pocket of structures obtained in presence of Tridecyl- Stigmatellin or NQNO implies a steric clash with the native position of the Phe40 aromatic ring. 5D. Picot, personal communication. 5D. Picot, personal communication. The Phe40 residue of subunit IV therefore stands as a key residue for the plasticity of the site, making it an ideal mutagenesis target when attempting to alter the possible interactions between ci and the quinone or quinol (4Kurisu G. Zhang H. Smith J.L. Cramer W.A. Science. 2003; 302: 1009-1014Crossref PubMed Scopus (596) Google Scholar, 5Stroebel D. Choquet Y. Popot J.L. Picot D. Nature. 2003; 426: 413-418Crossref PubMed Scopus (507) Google Scholar) (Fig. 1). Here we present the consequences of the substitution of Phe40 by a tyrosine on the properties and function of the ci heme. The H6F5 strain (mt+), expressing a cytochrome f version histidine tagged at its C-terminal end (21Choquet Y. Zito F. Wostrikoff K. Wollman F.A. Plant Cell. 2003; 15: 1443-1454Crossref PubMed Scopus (62) Google Scholar), and the ΔpetD (mt+) deletion strain (22Kuras R. Wollman F.A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar) were used as recipient strains in chloroplast transformation experiments. H6F5, ΔpetD, and mutant strains were grown on Tris-acetate-phosphate medium (pH 7.2) at 25 °C under dim light (5–6 μE·m−2·s−1) (23Harris E.H. The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego1989Google Scholar). The H6F5 strain was used as a wild type reference in all experiments. The pWQH6 plasmid, carrying the entire petD sequence, together with the C-terminal end of the sequence coding for histidine-tagged cytochrome f was obtained by ligation of the 0.55-kb EcoRV-AccI fragment recovered from plasmid pFWH6 (5Stroebel D. Choquet Y. Popot J.L. Picot D. Nature. 2003; 426: 413-418Crossref PubMed Scopus (507) Google Scholar) into the corresponding sites of plasmid pdWQ (21Choquet Y. Zito F. Wostrikoff K. Wollman F.A. Plant Cell. 2003; 15: 1443-1454Crossref PubMed Scopus (62) Google Scholar). The mutated version of the petD gene was created by PCR-mediated site-directed mutagenesis. Plasmid pdΔHI.I (22Kuras R. Wollman F.A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar), carrying the entire petD coding sequence was used as a template in PCRs performed with ArrowTM Taq DNA polymerase, employed according to the manufacturer's instructions, using the primers: DirF40Y (5′-GGGTGGCCAAACGATTTATTATACATGTACCCTGTTGTTATTTTAGGTACATTT-3′) and RevQi (5′-GGGTGGCCAAGCAGGTTCAC CGTAAGTGTT-3′), where nucleotides written in bold differ from the WT sequence. The PCR product was digested with MscI (a single restriction site underlined in the sequence of the primers) and religated onto itself to yield plasmid pdΔHF40Y. This plasmid was digested with HindIII and NcoI, and the resulting 833-bp fragment was ligated into the corresponding sites of plasmid pWQH6 to yield plasmid pdF40Y. Plasmid pdK-F40Y was constructed by introducing the 1.9-kb SmaI-EcoRV aadA cassette (24Goldschmidt-Clermont M. Nucleic Acids Res. 1991; 19: 4083-4089Crossref PubMed Scopus (399) Google Scholar), in the same orientation as the petD gene, into the EcoRV site of plasmid pdF40Y. The ΔpetD (22Kuras R. Wollman F.A. EMBO J. 1994; 13: 1019-1027Crossref PubMed Scopus (155) Google Scholar) and H6F5 (21Choquet Y. Zito F. Wostrikoff K. Wollman F.A. Plant Cell. 2003; 15: 1443-1454Crossref PubMed Scopus (62) Google Scholar) strains were transformed by tungsten particle bombardment according to Boynton et al. (25Boynton J.E. Gillham N.W. Harris E.H. Hosler J.P. Johnson A.M. Jones A.R. Randolph-Anderson B.L. Robertson D. Klein T.M. Shark K.B. Science. 1988; 240: 1534-1538Crossref PubMed Scopus (712) Google Scholar). When using plasmid pdF40Y to bombard the non-photosynthetic ΔpetD strain, transformed clones were selected on minimum medium at 60 μE·m−2·s−1. When using plasmid pdK-F40Y to bombard the H6F5 strain, transformants were selected on Tris-acetate-phosphate medium supplemented with spectinomycin (100 μg·ml−1). Transformed cells were subcloned several times on selective medium until they reach homoplasmy, assessed by restriction fragment length polymorphism analysis of specific PCR fragments (26de Lacroix de Lavalette A. Barbagallo R.P. Zito F. C. R. Biol. 2008; 331: 510-517Crossref PubMed Scopus (2) Google Scholar). Cells grown to a density of 4 × 106 ml−1 were broken in "bead beater" (Biospec-Products) according to the manufacturer's instructions. The membrane fraction was collected by centrifugation and resuspended in 10 mm Tricine, pH 8, at a chlorophyll concentration of 3 mg·ml−1. For SDS-PAGE, the membrane proteins were resuspended in 100 mm dithiothreitol and 100 mm Na2CO3 and solubilized by 2% SDS at 100 °C for 1 min. Polypeptides were separated on a 12–18% polyacrylamide gel containing 8 m urea. Immunoblotting was performed as described in Pierre et al. (27Pierre Y. Breyton C. Kramer D. Popot J.L. J. Biol. Chem. 1995; 49: 29342-29349Abstract Full Text Full Text PDF Scopus (147) Google Scholar). Cytochrome b6f complexes were isolated as described in Ref. 5Stroebel D. Choquet Y. Popot J.L. Picot D. Nature. 2003; 426: 413-418Crossref PubMed Scopus (507) Google Scholar and titrated accordingly to Alric et al. (14Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15860-15865Crossref PubMed Scopus (72) Google Scholar). In vitro electron transfer activity was measured as described in Ref. 27Pierre Y. Breyton C. Kramer D. Popot J.L. J. Biol. Chem. 1995; 49: 29342-29349Abstract Full Text Full Text PDF Scopus (147) Google Scholar. Cytochrome b6f complexes were analyzed by size exclusion chromatography in 20 mm Tris-HCl, pH 8.0, 250 mm NaCl, 0.2 mm C12M onto an exclusion Superdex 200 HR Amersham Biosciences column (28Huang D. Everly R.M. Cheng R.H. Heymann J.B. Schägger H. Sled V. Ohnishi T. Baker T.S. Cramer W.A. Biochemistry. 1994; 33: 4401-4409Crossref PubMed Scopus (104) Google Scholar). The phenotype of b6f mutants was characterized by their fluorescence induction kinetics. The measurements were performed at room temperature on a home-built fluorimeter, with continuous illumination at 520 nm and fluorescence detection in the far-red region (29Bennoun P. Delepelaire P. Edelman M. Chua N.H. Hallick R.B. Methods in Chloroplast Molecular Biology. Elsevier Biomedical Press, 1982: 25-38Google Scholar, 30Zito F. Kuras R. Choquet Y. Kössel H. Wollman F.A. Plant Mol. Biol. 1997; 33: 79-86Crossref PubMed Scopus (66) Google Scholar, 31Lemaire C. Girard-Bascou J. Wollman F.A. Biggins J. Progress in Photosynthesis Research. IV. Martinus Nijhoff Publishers, 1987: 655-658Google Scholar). Time-resolved light-induced absorbance changes in whole cells of C. reinhardtii were monitored with a pulsed differential LED spectrophotometer JTS 10 Bio-Logic, whereas redox-induced and CO-induced absorbance changes in purified b6f preparations were measured with a xenon flash lamp spectrophotometer, as previously described respectively by Joliot et al. (32Joliot P. Beal D. Frilley B. J. Chim. Phys. 1980; 77: 209-216Crossref Google Scholar) and in Alric et al. (14Alric J. Pierre Y. Picot D. Lavergne J. Rappaport F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15860-15865Crossref PubMed Scopus (72) Google Scholar). To characterize the kinetics of CO binding after laser flash photolysis, the purified b6f complex (5 μm, pH 8) was thoroughly degassed, then reduced by addition of dithionite, and then equilibrated with ∼1 atm of CO. Cytochrome b redox changes were measured in the presence of NQNO to obtain full inhibition of cytochrome b6 oxidation. NQNO was synthesized in the laboratory according to the procedure described in Ref. 33Cornforth J.W. James A.T. Biochem. J. 1956; 63: 124-130Crossref PubMed Scopus (73) Google Scholar. FOS-choline-14 (n-tetradecylphosphocholine) was purchased from Anatrace. We investigated by site-directed mutagenesis the importance of the accessibility to the heme ci by replacing the Phe40 of subunit IV, in van der Waal's contact with the plane of the ci, heme, by a tyrosine. Our first attempt to recover phototrophic clones following transformation of the ΔpetD strain with plasmid pdF40Y proved unsuccessful. As an alternative strategy, we used the histidine-tagged H6F5 strain as a recipient strain for transformation with the pd-KF40Y plasmid and recovered transformed clones on spectinomycin supplemented medium (24Goldschmidt-Clermont M. Nucleic Acids Res. 1991; 19: 4083-4089Crossref PubMed Scopus (399) Google Scholar). Five independent dK-F40Y clones, hereafter referred to as F40Y mutants, were brought to homoplasmy with respect to the petD mutation. Because transformation of the ΔpetD strain by the mutated copy of the petD gene failed to rescue phototrophic growth, we expected the recovered homoplasmic transformants to display fluorescence induction kinetics typical of mutants lacking cytochrome b6f activity (30Zito F. Kuras R. Choquet Y. Kössel H. Wollman F.A. Plant Mol. Biol. 1997; 33: 79-86Crossref PubMed Scopus (66) Google Scholar, 31Lemaire C. Girard-Bascou J. Wollman F.A. Biggins J. Progress in Photosynthesis Research. IV. Martinus Nijhoff Publishers, 1987: 655-658Google Scholar, 34Kuras R. de Vitry C. Choquet Y. Girard-Bascou J. Culler D. Büschlen S. Merchant S. Wollman F.A. J. Biol. Chem. 1997; 272: 32427-32435Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Surprisingly, they still displayed unimpaired fluorescence induction kinetics (Fig. 2) (26de Lacroix de Lavalette A. Barbagallo R.P. Zito F. C. R. Biol. 2008; 331: 510-517Crossref PubMed Scopus (2) Google Scholar). They grew as the wild type on minimum medium and did not show any photo-sensitivity under high light (200 μE·m−2·s−1), neither in photoautotrophic nor in mixotrophic conditions (data not shown). On this basis, we expected the electron transfer chain to be active and assessed its function by time-resolved absorption spectroscopy. Fig. 3A shows the slow electrogenic phase associated with the turnover of the b6f complex in WT and mutant strains. At 100 μs the absorption changes result from the charge separation event at the level of PSI; the subsequent electrogenic phase that develops with a ∼2-ms half-time witnesses the cytochrome b6f turnover (35Joliot P. Delosme R. Biochim. Biophys. Acta. 1974; 357: 267-284Crossref PubMed Scopus (159) Google Scholar, 36Junge W. Witt H.T. Z. Naturforsch. B. 1968; 23: 244-254Crossref PubMed Scopus (338) Google Scholar). In the F40Y mutant, the amplitude of this phase was dramatically decreased. We then measured the kinetics of the absorption changes associated with the redox changes of the b hemes, at 563 nm (Fig. 3B). In the WT, in the absence of inhibitors, a transient reduction (upward signal) is followed by a pronounced oxidation phase (downward signal). The negative signal observed 1 s after a flash shows that the overall reaction results in the net oxidation of a b heme. Thus, in these experimental conditions, the bH and bL hemes were respectively reduced and oxidized in the dark, so that the injection of one electron into the low potential chain allowed the reduction of a quinone at the Qi site and the associated oxidation of the two b hemes. The addition of NQNO (50 μm) slows down this latter reaction and, accordingly, allows the observation of the full extent of the b heme reduction phase (37Selak M.A. Whitmarsh J. Photochem. Photobiol. 1984; 39: 485-489Crossref Scopus (19) Google Scholar). In the mutant, in the absence of NQNO, we observed a pronounced reduction of b heme with a half-time of ∼2 ms similar to the one observed in the WT in the presence of NQNO, followed by a very slow oxidation phase. The addition of NQNO had no further effect on this oxidation phase. This result, together with the decreased amplitude of the electrogenic phase, indicates that in the mutant, under the conditions of the experiments, the bH heme was likely reduced in the dark, whereas the bL heme was oxidized and that quinol oxidation at the Qo site operated normally. The dramatic slow down of the oxidation of the b hemes indicates that quinone reduction at the Qi site is strongly affected, consistent with the quasi absence of electrogenic phase. The F40Y mutation thus seems to cause a strong inhibition of the Qi site. Yet contrasting results were obtained when studying the turnover of the b6f complex under continuous illumination. As shown above, the fluorescence induction curves of F40Y cells are virtually indistinguishable from that commonly obtained with WT. Fig. 2 shows typical measurements carried out under aerobic conditions in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Surprisingly, in the F40Y mutant, the steady state fluorescence level, reached after a few seconds of illumination, was similar to the WT one. Thus, despite the apparent loss of Qi site activity, as observed under single turnover flash experiments, the electron flows through the cytochrome b6f complex are similar in both strains. It is of note however that the fluorescence measurements were performed under aerobic conditions (oxidized PQ pool), whereas the flash-induced kinetics were obtained under anaerobic conditions (reduced PQH2 pool), leaving open the possibility of a different occupancy of the Qi site by its substrate in the two experiments. We thus measured the overall activity of the photosynthetic chain during illumination under saturating light intensity, in aerobic as well as anaerobic conditions. In such conditions, the limiting step of the photosynthetic electron flow bears on the cytochrome b6f complex. Electrochromic changes of carotenoids, measured at 520 nm, were used as a probe of the transmembrane potential (38Joliot P. Joliot A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10209-10214Crossref PubMed Scopus (286) Google Scholar). As shown in Fig. 4 the time course of the absorption changes encompasses a very fast rise (with an initial slope of 1000 charges translocated per second), because of the charge separation at the level of the photosynthetic chain (PSII-PQ-b6f-plastocyanin-PSI), followed by a decay likely reflecting the activation of CF0-CF1 ATP synthase. After ∼1 s of illumination, the membrane potential reaches a quasi-steady state regime where the generation of the transmembrane electric field by the activity of the photosynthetic chain nearly compensates its dissipation by the ATP synthase. When the light is switched off, the photochemical process is instantaneously stopped, and the absorption changes decay as the transmembrane potential is rapidly consumed by the CF0-CF1 ATP synthase. The difference between the slopes of the signal before and after the light is switched off is indicative of the photochemical activity (38Joliot P. Joliot A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 10209-10214Crossref PubMed Scopus (286) Google Scholar, 39Sacksteder C.A. Kramer D.M. Photosynth. Res. 2000; 66: 145-158Crossref PubMed Scopus (122) Google Scholar). We first validated this approach by the study of a b6f lacking mutant (Fig. 4B). Switching off the illumination did not induce any variation of the slope of the electrochromic band shift after 1 s of illumination. In the WT and in the F40Y mutant (Fig. 4, A and C), under aerobic and anaerobic conditions, a clear change in the slope was observed when the light was switched off, showing that, under illumination, the consumption of the transmembrane potential by the ATP synthase is compensated by its photo-induced formation. In the WT, after normalization of the change in slope by the signal induced by a single turnover flash (which yields the absorption changes resulting from the transfer of 2 electrons across the membrane), we estimated the photochemical activity to ∼100 e−·s−1 (aerobic) and 40 e−·s−1 (anaerobic). In the mutant, these rates were estimated to 70 and 40 charges/s, respectively. At this stage we are thus confronted to an apparent paradox. On the one hand, the quinone reduction activity of the Qi site is strongly impaired as observed in the flash experiments. On the other hand, the overall activity of the mutated b6f complex only slightly differs from the WT one under continuous illumination, suggesting that the functional constraints underlying the blockage of the Qi site function under single flash conditions are relieved in a continuous illumination regime. To obtain a more complete picture of the interaction between the Tyr side chain and the ci heme, we studied the physico-chemical properties of the ci heme in purified b6f complex from the mutant. The mutant accumulated the major subunits of the cytochrome b6f complex at approximately wild type levels (Fig. 5A). In particular the content in subunit IV, which harbors the mutations, was unaltered. To further identify the ci heme associated with cyt b6, the gels were stained with 3,3′,5,5′ tetramethylbenzidine, a specific staining for hemes (40de Vitry C. Desbois A. Redeker V. Zito F. Wollman F.A. Biochemistry. 2004; 43: 3956-3968Crossref PubMed Scopus (37) Google Scholar). The peroxidase activity associated with cyt b6 proved to be SDS-resistant consistent with the covalent binding of a heme to this subunit, strongly supporting the presence of the ci heme. The oligomerization state of the cytochrome b6f complex, as measured by size exclusion chromatography, was not affected by the mutation (Fig. 5B). The b6f complex purified from the mutant was then tested for its ability to mediate the transfer of electrons between plastoquinol and oxidized plastocyanin in vitro. Its activity (∼350 e−·f−1·s−1) was found similar to that of the wild type complex (∼400 e−·f−1·s−1). This is satisfyingly consistent with the conclusion drawn from the in vivo activity, described above, that the turnover rate of the mutated complex hardly differs from the WT one. The substitution of the Phe40 by a Tyr has no effect on the midpoint potentials of the bL and bH hemes but strongly modifies the Em of heme ci (Fig. 6B), which was downshifted from +100 mV in the WT to −200 mV in the mutant (at pH 7.0). As for the WT (Fig. 6A), the midpoint potential was pH-dependent with a slope of −60 mV/pH unit (Fig. 6B). The F40Y mutation also affected the (oxidized − reduced) spectrum of the ci heme (Fig. 7A), which displays a shoulder around 440 nm and an overshoot at 380 nm. These features were even more pronounced after the additi
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