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PubMed2000

On the spatial organization of hemes and chlorophyll in cytochrome b(6)f. A linear and circular dichroism study.

The organization of chromophores in the cytochrome b(6) f from Chlamydomonas reinhardtii has been studied spectroscopically. Linear dichroism (LD) measurements, performed on the complex co-reconstituted into vesicles with photosynthetic reaction centers as an internal standard, allow the determination of the orientations of the chromophore with respect to the membrane plane. The orientations of the b(H)- and b(L)-hemes are comparable to those determined crystallographically on the cytochrome bc(1). The excitonic CD signal, resulting from the interaction between b-hemes, is similar to that reported for the cytochrome bc(1). LD and CD data are consistent with the differences between the b(6) f and bc(1) leaving the orientation of the b-hemes unaffected. By contrast, the LD data yield a different orientation for the heme f as compared either to the heme c(1) in the crystallographic structures or to the heme f as studied by electron paramagnetic resonance. This difference could either result from incorrect assumptions regarding the orientations of the electronic transitions of the f-heme or may point to the possibility of a redox-dependent movement of cytochrome f. The chlorophyll a was observed in a well defined orientation, further corroborating a specific binding site for it in the b(6) f complex.

Animals↗
PubMed2006

Structure of cytochrome c6A, a novel dithio-cytochrome of Arabidopsis thaliana, and its reactivity with plastocyanin: implications for function.

Cytochrome c6A is a unique dithio-cytochrome present in land plants and some green algae. Its sequence and occurrence in the thylakoid lumen suggest that it is derived from cytochrome c6, which functions in photosynthetic electron transfer between the cytochrome b6f complex and photosystem I. Its known properties, however, and a strong indication that the disulfide group is not purely structural, indicate that it has a different, unidentified function. To help in the elucidation of this function the crystal structure of cytochrome c6A from Arabidopsis thaliana has been determined in the two redox states of the heme group, at resolutions of 1.2 A (ferric) and 1.4 A (ferrous). These two structures were virtually identical, leading to the functionally important conclusion that the heme and disulfide groups do not communicate by conformational change. They also show, however, that electron transfer between the reduced disulfide and the heme is feasible. We therefore suggest that the role of cytochrome c6A is to use its disulfide group to oxidize dithiol/disulfide groups of other proteins of the thylakoid lumen, followed by internal electron transfer from the dithiol to the heme, and re-oxidation of the heme by another thylakoid oxidant. Consistent with this model, we found a rapid electron transfer between ferro-cytochrome c6A and plastocyanin, with a second-order rate constant, k2=1.2 x 10(7) M(-1) s(-1).

Amino Acid Sequence↗
PubMed2001

Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b(6) in Synechococcus sp. PCC 7002.

The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b(6) subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b(6) at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc(1) complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc(1) complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc(1) complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure-function relationships in the cytochrome bf complex.

Alleles↗
PubMed1986

Spatial organization of the cytochrome b6-f complex within chloroplast thylakoid membranes.

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b6-f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3',5,5'-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b6-f antibodies; (4) agglutination of membrane fragments with anti-Cyt b6-f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b6-f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b6-f throughout all thylakoid membranes.

Chloroplasts↗
PubMed1995

Maturation of pre-apocytochrome f in vivo. A site-directed mutagenesis study in Chlamydomonas reinhardtii.

The biosynthesis of cytochrome f is a multistep process which requires processing of the precursor protein and covalent ligation of a c-heme upon membrane insertion of the protein. The crystal structure of a soluble form of cytochrome f has revealed that one axial ligand of the c-heme is provided by the alpha-amino group of Tyr1 generated upon cleavage of the signal sequence from the precursor protein (Martinez S. E., Huang D., Szczepaniak A., Cramer W.A., and Smith J. L. (1994) Structure 2, 95-105). We therefore investigated, by site-directed mutagenesis, the possible interplay between protein processing and heme attachment to cytochrome f in Chlamydomonas reinhardtii. These modifications were performed by chloroplast transformation using a petA gene encoding the full-length precursor protein and also a truncated version lacking the C-terminal membrane anchor. We first substituted the two cysteinyl residues responsible for covalent ligation of the c-heme, by a valine and a leucine, and showed that heme binding is not a prerequisite for cytochrome f processing. In another series of experiments, we replaced the consensus cleavage site for the thylakoid processing peptidase, AQA, by an LQL sequence. The resulting transformants were nonphototrophic and displayed delayed processing of the precursor form of cytochrome f, but nonetheless both the precursor and processed forms showed heme binding and assembled in cytochrome b6f complexes. Thus, pre-apocytochrome f adopts a suitable conformation for the cysteinyl residues to be substrates of the heme lyase and pre-holocytochrome f folds in an assembly-competent conformation. In the last series of experiments, we compared the rates of synthesis and degradation of the various forms of cytochrome f in the four types of transformants under study: (i) the C terminus membrane anchor apparently down-regulates the rate of synthesis of cytochrome f and (ii) degradation of misfolded forms of cytochrome f occurs by a proteolytic system intimately associated with the thylakoid membranes.

Amino Acid Sequence↗
PubMed1985

Preparation and reconstitution of a phospholipid deficient cytochrome b6-f complex from spinach chloroplasts.

A simple, rapid procedure suitable for large scale preparation of a lipid deficient cytochrome b6-f complex from spinach chloroplasts has been developed. The procedure involves solubilization with a mixture of sodium cholate and octylglucoside, ammonium sulfate fractionation and calcium phosphate column chromatography. The purified complex contains, in nanomoles per milligram protein, 20.6 cytochrome b, 10.8 cytochrome f and 54 phospholipids. The purified complex has little plastoquinol-cytochrome c reductase activity in the absence of added lipid. Full reductase activity was reconstituted by the addition of plastoquinone prior to the addition of lipid.

Chloroplasts↗
PubMed1985

Topographical studies of the polypeptide subunits of the thylakoid cytochrome b6-f complex.

The orientation of specific polypeptides of the cytochrome b6-f complex with respect to the chloroplast stromal phase has been studied using trinitrobenzenesulfonate (TNBS) and pronase E as impermeant modifying reagents. Of the four polypeptides of the complex (33,23,20 and 17 kDa), only cytochrome f was labeled by 14C-TNBS in unfractionated membranes. However, to a varying degree, all of the constituent polypeptides were sensitive to pronase digestion and, in the case of cytochrome f, it was possible, by immunoblotting techniques to identify several degradation products. These results are discussed in relation to the organization of the cytochrome complex in thylakoid membranes and argue for an exposure to the stromal phase of all of the polypeptides, while functional considerations indicate that at least cytochrome f and the Rieske iron-sulfur protein have a possible transmembrane organization.

Animals↗
PubMed1991

DCCD binds to cytochrome b6 of a cytochrome bf complex isolated from spinach chloroplasts and inhibits proton translocation.

Radiolabeled N,N'-dicyclohexylcarbodiimide (DCCD) was bound selectively in a time- and concentration-dependent manner to cytochrome b6 of an enzymatically active cytochrome bf complex isolated from spinach chloroplasts. Maximum labeling of cytochrome b6 was observed with 30 nmol DCCD per nmol cytochrome b6 in the cytochrome bf complex incubated for 30-60 min at 12 degrees C. After incubation of the cytochrome bf complex with DCCD under these conditions, the rate of proton ejection in the complex reconstituted into liposomes was decreased approximately 65-70% when compared to controls; however, under these same conditions the rate of electron transfer through either the soluble bf complex or the complex reconstituted into liposomes was only decreased around 20%. These results suggest that the mechanism of proton translocation through the cytochrome bf complex of spinach chloroplasts is similar to that of the cytochrome bc1 complex from yeast mitochondria in which proton pumping but not electron transfer is also inhibited by DCCD (D. S. Beattie and A. Villalobo, 1982, J. Biol. Chem. 257, 14,745-14,752).

Amino Acid Sequence↗
PubMed1997

Analysis of the interaction of water with the manganese cluster of photosystem II using isotopically labeled water.

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.

Deuterium Oxide↗
PubMed1992

Further identification of the exoplasmic face particles on the freeze-fractured thylakoid membranes: a study using double and triple mutants from Chlamydomonas reinhardtii lacking various photosystem II subunits and the cytochrome b6/f complex.

About 20% of the exoplasmic face (EF) particles present in the freeze-fractured thylakoid membranes of the wild type strain of Chlamydomonas reinhardtii remain in mutants lacking photosystem II (PSII) because of the absence of either one of the two PSII subcomplexes CP43 or D1/D2/CP47. We show that about half of these residual EF particles can be accounted for by PSII subcomplexes still present in such mutants, and by cytochrome (cyt) b6/f complexes. Analysis of double mutants lacking both types of protein complexes points to an association of cyt b6/f complexes with PSII subcomplexes in some of these EF particles and to a requirement in cyt b6/f complexes for the translocation of each of the two PSII subcomplexes (the CP43 subunit and the D1/D2/CP47 subcomplex) from the unstacked to the stacked regions of the thylakoid membranes.

Animals↗
PubMed1991

Nucleotide sequences of the continuous and separated petA, petB and petD chloroplast genes in Chlamydomonas reinhardtii.

We have mapped and sequenced the petA (cytf), petB (cytb6) and petD (subunit IV) genes on the chloroplast genome of Chlamydomonas reinhardtii. At variance with the pet genes in higher plant chloroplasts, the petB and petD genes are continuous, not adjacent and not located next to the psbB gene. The corresponding polypeptide sequences are highly conserved when compared with their counterparts from other sources but have a few features specific of algal cytb6/f complexes. In particular the transit sequence of cytf displays unique characteristics when compared with those previously described for cytf in higher plants.

Amino Acid Sequence↗
PubMed1992

Binding of dicyclohexylcarbodiimide to aspartate-155 or glutamate-166 of cytochrome b6 in a cytochrome bf complex isolated from spinach thylakoids.

In a recent study [Wang & Beattie (1991) Arch. Biochem. Biophys. 291, 363-370], we reported that dicyclohexylcarbodiimide (DCCD) inhibited proton translocation in the cytochrome bf complex reconstituted into proteoliposomes and was bound selectively to cytochrome b6. To establish the site of binding of DCCD on cytochrome b6, the cytochrome bf complex labeled with [14C]DCCD was selectively digested with chymotrypsin and trypsin. A 17-kDa fragment containing radioactive DCCD and the heme moiety was obtained after chymotrypsin digestion, while a 12.5-kDa fragment containing both radioactive DCCD and the heme moiety was obtained after trypsin digestion, suggesting that the site of DCCD binding might be on aspartate-140, aspartate-155, or glutamate-166. Extensive digestion of cytochrome b6 isolated from a [14C]DCCD-labeled cytochrome bf complex with trypsin followed by isolation and sequencing of two radioactive peptides obtained revealed that DCCD is bound at either residue aspartate-155 or residue glutamate-166 localized in amphipathic extramembranous helix IV. In addition, the cytochrome bf complex labeled with [14C]DCCD was reconstituted into liposomes and digested with trypsin. Three fragments of 9.3, 10.5, and 11.5 kDa were obtained, suggesting that the four-helix model for the topography of cytochrome b6 in the membrane is correct.

Amino Acid Sequence↗
PubMed2002

Electron transport routes in whole cells of Synechocystis sp. strain PCC 6803: the role of the cytochrome bd-type oxidase.

The plastoquinone pool is the central switching point of both respiratory and photosynthetic electron transport in cyanobacteria. Its redox state can be monitored noninvasively in whole cells using chlorophyll fluorescence induction, avoiding possible artifacts associated with thylakoid membrane preparations. This method was applied to cells of Synechocystis sp. PCC 6803 to study respiratory reactions involving the plastoquinone pool. The role of the respiratory oxidases known from the genomic sequence of Synechocystis sp. PCC 6803 was investigated by a combined strategy using inhibitors and deletion strains that lack one or more of these oxidases. The putative quinol oxidase of the cytochrome bd-type was shown to participate in electron transport in thylakoid membranes. The activity of this enzyme in thylakoids was strongly dependent on culture conditions; it was increased under conditions where the activity of the cytochrome b(6)f complex alone may be insufficient for preventing over-reduction of the PQ pool. In contrast, no indication of quinol oxidase activity in thylakoids was found for a second alternative oxidase encoded by the ctaII genes.

Cyanobacteria↗
PubMed1985

Characterization of a photosynthetic mutant of Lemna lacking the cytochrome b6-f complex.

A photosynthetic mutant of Lemna perpusilla (no. 1073) has been examined by spectrophotometric and immunoblotting techniques in order to localize the site of defect. In contrast to previous conclusions (Shahak, Y., Posner, H.B. and Avron, M. (1976) Plant Physiol. 57, 577-679), neither cytochrome f nor cytochrome b6 could be detected spectrophotometrically in the mutant. Furthermore, immunoblotting using antibodies specific for each of the four constituent subunits of the cytochrome b6-f complex demonstrate that the entire complex is absent in the mutant. The light-harvesting chlorophyll-protein complex of Photosystem II is present in similar amounts in wild-type and mutant Lemna. However, the total amount of plastoquinone-9 is reduced by approx. 65% in the mutant strain, while the photoreducible plastoquinone-9 pool is comparable in wild-type and mutant Lemna.

Antibodies↗
PubMed2001

Assembly of cytochrome f into the cytochrome bf complex in isolated pea chloroplasts.

Structural features of cytochrome f necessary for assembly into the cytochrome bf complex were examined in isolated pea chloroplasts following import of (35)S-labelled chimeric precursor proteins, consisting of the presequence of the small subunit of Rubisco fused to the turnip cytochrome f precursor. Assembly was detected by nondenaturing gel electrophoresis of dodecyl maltoside-solubilized thylakoid membranes. A cytochrome f polypeptide unable to bind haem because of mutagenesis of Cys21 and Cys24 to alanine residues was assembled into the complex and had similar stability to the wild-type polypeptide. This indicates that covalent haem binding to cytochrome f is not necessary for assembly of the protein into the cytochrome bf complex. A truncated protein lacking the C-terminal 33 amino acid residues, including the transmembrane span and the stroma-exposed region, was translocated across the thylakoid membrane, had a similar stability to wild-type cytochrome f but was not assembled into the complex. This indicates that the C-terminal region of cytochrome f is important for assembly into the complex. A mutant cytochrome f unable to bind haem and lacking the C-terminal region was also translocated across the thylakoid membrane but was extremely labile, indicating that, in the absence of the C-terminal membrane anchor, haem-less cytochrome f is recognized by a thylakoid proteolytic system.

Alanine↗
PubMed2005

Cytochrome b6 arginine 214 of Synechococcus sp. PCC 7002, a key residue for quinone-reductase site function and turnover of the cytochrome bf complex.

Quinone-reductase (Q(i)) domains of cyanobacterial/chloroplast cytochrome bf and bacterial/mitochondrial bc complexes differ markedly, and the cytochrome bf Q(i) site mechanism remains largely enigmatic. To investigate the bf Q(i) domain, we constructed the mutation R214H, which substitutes histidine for a conserved arginine in the cytochrome b(6) polypeptide of the cyanobacterium Synechococcus sp. SPCC 7002. At high light intensity, the R214H mutant grew approximately 2.5-fold more slowly than the wild type. Slower growth arose from correspondingly slower overall turnover of the bf complex. Specifically, as shown in single flash turnover experiments of cytochrome b(6) reduction and oxidation, the R214H mutation partially blocked electron transfer to the Q(i) site, mimicking the effect of the Q(i) site inhibitor 2-N-4-hydroxyquinoline-N-oxide. The kinetics of cytochrome b(6) oxidation were largely unaffected by hydrogen-deuterium exchange in the mutant but were slowed considerably in the wild type. This suggests that although protonation events influenced the kinetics of cytochrome b(6) oxidation at the Q(i) site in the wild type, electron flow limited this reaction in the R214H mutant. Redox titration of membranes revealed midpoint potentials (E(m,7)) of the two b hemes similar to those in the wild type. Our data define cytochrome b(6) Arg(214) as a key residue for Q(i) site catalysis and turnover of the cytochrome bf complex. In the recent cytochrome bf structures, Arg(214) lies near the Q(i) pocket and the newly discovered c(i) or x heme. We propose a model for Q(i) site function and a role for Arg(214) in plastoquinone binding.

Alleles↗
PubMed1994

The assembly of cytochrome b6/f complexes: an approach using genetic transformation of the green alga Chlamydomonas reinhardtii.

As an approach to the study of the biogenesis of the cytochrome b6/f complex, we characterized the behaviour of its constitutive subunits in mutant strains of Chlamydomonas reinhardtii bearing well-defined mutations. To this end, we have constructed three deletion mutant strains, each lacking one of the major chloroplast pet genes: the delta petA, delta petB and delta petD strains were unable to synthesize cyt f, cyt b6 and subunit IV (suIV) respectively. Western blotting analysis, pulse-labelling and pulse-chase experiments allowed us to compare the cellular accumulation, the rates of synthesis and the turnover of the cyt b6/f subunits remaining in the various strains. We show that the rates of synthesis of cyt b6 and suIV are independent of the presence of the other subunits of the complex but that their stabilization in the thylakoid membranes is a concerted process, with a marked dependence of suIV stability on the presence of cyt b6. In contrast, mature cyt f was stable in the absence of either suIV or cyt b6 but its rate of synthesis was severely decreased in these conditions. We conclude that the stoichiometric accumulation of the chloroplast-encoded subunits of the cyt b6/f complex results from two regulation processes: a post-translational regulation leading to the proteolytic disposal of unassembled cyt b6 and suIV and a co-translational (or early post-translational) regulation which ensures the production of cyt f next to its site of assembly.

Animals↗
PubMed1996

Some characteristics of cytochrome f in the cyanobacterium Phormidium laminosum: its sequence and charge properties in the reaction with plastocyanin.

Part of the petCA operon was cloned and the sequence of the cytochrome f gene from the moderately thermophilic cyanobacterium Phormidium laminosum determined. A partial sequence of the petC gene encoding the Rieske iron-sulphur protein was also obtained. The cytochrome f gene encodes a mature protein of 385 residues and a leader sequence of 45 residues. The mature protein contains several acidic or neutral residues corresponding to basic residues in the turnip protein. Some of the latter are thought to be important for the interaction with plastocyanin via its "eastern' face. Many of the corresponding residues on the eastern face of P. laminosum plastocyanin are either basic or neutral instead of acidic. These comparisons suggested that the local charges on P. laminosum cytochrome f that are important for its interaction with the homologous plastocyanin may be negative rather than positive. The importance of acidic groups was confirmed by measuring the rates of reduction of horse heart cytochrome c and P. laminosum and spinach plastocyanins by the cytochrome bf complex isolated from P. laminosum. P. laminosum plastocyanin gave the highest rates, which decreased at high ionic strength, confirming the importance of positive local charges on this protein. When extrapolated to infinite ionic strength the rates observed with the two kinds of plastocyanin were similar, but cytochrome c became unreactive. An optimum was observed in the ionic strength response with P. laminosum plastocyanin.

Amino Acid Sequence↗