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PubMed2003

An atypical haem in the cytochrome b(6)f complex.

Photosystems I and II (PSI and II) are reaction centres that capture light energy in order to drive oxygenic photosynthesis; however, they can only do so by interacting with the multisubunit cytochrome b(6)f complex. This complex receives electrons from PSII and passes them to PSI, pumping protons across the membrane and powering the Q-cycle. Unlike the mitochondrial and bacterial homologue cytochrome bc(1), cytochrome b(6)f can switch to a cyclic mode of electron transfer around PSI using an unknown pathway. Here we present the X-ray structure at 3.1 A of cytochrome b(6)f from the alga Chlamydomonas reinhardtii. The structure bears similarities to cytochrome bc(1) but also exhibits some unique features, such as binding chlorophyll, beta-carotene and an unexpected haem sharing a quinone site. This haem is atypical as it is covalently bound by one thioether linkage and has no axial amino acid ligand. This haem may be the missing link in oxygenic photosynthesis.

Animals↗
PubMed2001

Photoinhibition of Chlamydomonas reinhardtii in State 1 and State 2: damages to the photosynthetic apparatus under linear and cyclic electron flow.

The relationship between state transitions and photoinhibition has been studied in Chlamydomonas reinhardtii cells. In State 2, photosystem II activity was more inhibited by light than in State 1. In State 2, however, the D1 subunit was not degraded, whereas a substantial degradation was observed in State 1. These results suggest that photoinhibition occurs via the generation of an intermediate state in which photosystem II is inactive but the D1 protein is still intact. The accumulation of this state is enhanced in State 2, because in this State only cyclic photosynthetic electron transport is active, whereas there is no electron flow between photosystem II and the cytochrome b(6)f complex (Finazzi, G., Furia, A., Barbagallo, R. P., and Forti, G. (1999) Biochim. Biophys. Acta 1413, 117-129). The activity of photosystem I and of cytochrome b(6)f as well as the coupling of thylakoid membranes was not affected by illumination under the same conditions. This allows repairing the damages to photosystem II thanks to cell capacity to maintain a high rate of ATP synthesis (via photosystem I-driven cyclic electron flow). This capacity might represent an important physiological tool in protecting the photosynthetic apparatus from excess of light as well as from other a-biotic stress conditions.

Adenosine Triphosphate↗
PubMed2000

Inhibition of electron transport at the cytochrome b(6)f complex protects photosystem II from photoinhibition.

Photoinhibition of photosystem II (PS II) activity was studied in thylakoid membranes illuminated in the presence of the inhibitor of the cytochrome b(6)f complex 2'iodo-6-isopropyl-3-methyl-2',4, 4'-trinitrodiphenylether (DNP-INT). DNP-INT was found to decrease photoinhibition. In the absence of DNP-INT, anaerobosis, superoxide dismutase and catalase protected against photoinhibition. No effect of these treatments was observed in the presence of DNP-INT. These data demonstrate that photoinhibition under these conditions is caused by reactive oxygen species which are formed most probably by the reduction of oxygen at photosystem I. The results are discussed in terms of the importance of photosynthetic control in protection against photoinhibition in vivo.

Anaerobiosis↗
PubMed2005

Higher plant-like fluorescence induction and thermoluminescence characteristics in cyanobacterium, Spirulina mutant defective in PQH2 oxidation by cytb6/f complex.

Characterization of the photosynthetic electron transport in a mutant of Spirulina platensis, generated by chemical mutagenesis, demonstrated that the electron transfer from the plastoquinone (PQ) to cytochrome b6/f was slowed. Thermoluminescence (TL) measurements suggested the presence of reversed energy flow via PQ, which resulted in an emergence of the plant-like after-glow TL band at 45 degrees C that could be enhanced by the transthylakoidal pH gradient and could be eliminated by an uncoupler, FCCP. The localization of the changes in the electron transport of the mutant cells measured by various methods revealed that the re-oxidation of the PQ pool is hampered in the mutant compared to the wild-type cells. The reduction in energy migration was localized between PQ and PS I reaction centers.

Cyanobacteria↗
PubMed1993

Further characterization of the psbH locus of Synechocystis sp. PCC 6803: inactivation of psbH impairs QA to QB electron transport in photosystem 2.

The psbH gene encodes a small protein which copurifies with photosystem 2. In the cyanobacterium Synechocystis sp. PCC 6803, psbH is located upstream of the cytochrome b6-f complex genes petC and petA. In striking contrast, in the genomes of plant chloroplasts, psbH is cotranscribed with petB and petD, encoding the other two major subunits of the cytochrome b6-f complex. We report that in Synechocystis sp. PCC 6803 monocistronic psbH and dicistronic petCA transcripts are probably initiated separately, each from DNA regions bearing some similarity to Escherichia coli sigma 70 promoters. Synechocystis sp. PCC 6803 psbH null mutants were generated by cartridge mutagenesis. Studies using a rapid screening procedure involving in situ complementation showed that the PsbH protein is not absolutely required for the assembly of a functionally active photosystem 2 complex since psbH insertion and deletion strains were able to grow photoautotrophically. The rate of photoautotrophic growth was, however, slower than the wild type, and studies of oxygen evolution, chlorophyll fluorescence, and thermoluminescence indicated that this reduction in growth rate is probably due mainly to an impairment in electron flow from QA to QB. We conclude, therefore, that the PsbH protein is not an absolute requirement for photosystem 2 activity but that it functions to optimize electron flow between the two secondary plastoquinone acceptors by interacting with the QB site on the D1 protein.

Base Sequence↗
PubMed2003

Knock-out of the genes coding for the Rieske protein and the ATP-synthase delta-subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression.

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b(6)/f (cyt b(6)/f) complex and the delta-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b(6)/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor Q(A) in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-delta destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b(6)/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

Arabidopsis↗
PubMed2001

Deuterium kinetic isotope effects in the p-side pathway for quinol oxidation by the cytochrome b(6)f complex.

Plastoquinol oxidation and proton transfer by the cytochrome b(6) f complex on the lumen side of the chloroplast thylakoid membrane are mediated by high and low potential electron transport chains. The rate constant for reduction, k(bred), of cytochrome b(6) in the low potential chain at ambient pH 7.5-8 was twice that, k(fred), of cytochrome f in the high potential chain, as previously reported. k(bred) and k(fred) have a similar pH dependence in the presence of nigericin/nonactin, decreasing by factors of 2.5 and 4, respectively, from pH 8 to an ambient pH = 6, close to the lumen pH under conditions of steady-state photosynthesis. A substantial kinetic isotope effect, k(H2O)/k(D2O), was found over the pH range 6-8 for the reduction of cytochromes b(6) and f, and for the electrochromic band shift associated with charge transfer across the b(6)f complex, showing that isotope exchange affects the pK values linked to rate-limiting steps of proton transfer. The kinetic isotope effect, k(bred)(H2O)/k(bred) (D2O) approximately 3, for reduction of cytochrome b in the low potential chain was approximately constant from pH 6-8. However, the isotope effect for reduction of cytochrome f in the high potential chain undergoes a pH-dependent transition below pH 6.5 and increased 2-fold in the physiological region of the lumen pH, pH 5.7-6.3, where k(fred)(H2O)/k(fred)(D2O) approximately 4. It is proposed that a rate-limiting step for proton transfer in the high potential chain resides in the conserved, buried, and extended water chain of cytochrome f, which provides the exit port for transfer of the second proton derived from p-side quinol oxidation and a "dielectric well" for charge balance.

Chloroplasts↗
PubMed1999

Effects of inhibitors on the activity of the cytochrome b(6)f complex: evidence for the existence of two binding pockets in the lumenal site.

The effects of two inhibitors of electron transfer in the cytochrome b(6)f complex have been studied in whole cells of Chlamydomonas reinhardtii. DNP-INT affected equally the two steps of the concerted oxidation of plastoquinol at the Q(o) site; it decreased the rates of both cytochrome f reduction and cytochrome b(6) turnover, without affecting the amplitude of their redox signals. On the contrary, DBMIB inhibited only the rate of cytochrome f reduction while reducing, at the same time, the amplitude of cytochrome b(6) signals. The accessibility of DNP-INT to the Q(o) site was unaffected by preillumination, while that of DBMIB was greatly enhanced, even after a single turnover of the cytochrome b(6)f complex. Similar results were obtained with a mutant strain (FUD2) where the Q(o) site has an affinity for plastoquinol that is diminished by a factor of approximately 50 [Finazzi, G., et al. (1997) Biochemistry 36, 2867-2874]. However, the binding of the two inhibitors was differentially influenced by the mutation: a factor of approximately 250 was calculated for DNP-INT and a factor of only approximately 5 for DBMIB. This suggests that they bind within the Q(o) site in two distinct pockets, which are differentially involved in the process of quinol oxidation, in agreement with a recent model where two distinct positions for the reduced and semireduced quinones are considered [Crofts, A. R., and Berry, E. A. (1998) Curr. Opin. Struct. Biol. 8, 501-509].

Animals↗
PubMed1999

Analysis of the nucleus-encoded and chloroplast-targeted rieske protein by classic and site-directed mutagenesis of Chlamydomonas.

Three mutants of the alga Chlamydomonas reinhardtii affected in the nuclear PETC gene encoding the Rieske iron-sulfur protein 2Fe-2S subunit of the chloroplast cytochrome b(6)f complex have been characterized. One has a stable deletion that eliminates the protein; two others carry substitutions Y87D and W163R that result in low accumulation of the protein. Attenuated expression of the stromal protease ClpP increases accumulation and assembly into b(6)f complexes of the Y87D and W163R mutant Rieske proteins in quantities sufficient for analysis. Electron-transfer kinetics of these complexes were 10- to 20-fold slower than those for the wild type. The deletion mutant was used as a recipient for site-directed mutant petC alleles. Six glycine residues were replaced by alanine residues (6G6A) in the flexible hinge that is critical for domain movement; substitutions were created near the 2Fe-2S cluster (S128 and W163); and seven C-terminal residues were deleted (G171och). Although the 6G6A and G171och mutations affect highly conserved segments in the chloroplast Rieske protein, photosynthesis in the mutants was similar to that of the wild type. These results establish the basis for mutational analysis of the nuclear-encoded and chloroplast-targeted Rieske protein of photosynthesis.

Amino Acid Sequence↗
PubMed1998

Inversions in the Chlamydomonas chloroplast genome suppress a petD 5' untranslated region deletion by creating functional chimeric mRNAs.

FUD6 is a non-photosynthetic Chlamydomonas mutant that lacks the cytochrome b6/f complex, due to a 236 bp deletion that removes the promoter and part of the 5' untranslated region (UTR) of the chloroplast petD gene, which encodes subunit IV of the complex. Two photosynthetic revertants of FUD6 that synthesized wild-type levels of subunit IV were found to contain related inversions of the chloroplast genome that resulted from recombination between small inverted repeats. These inversions created a functional chimeric petD gene that includes the promoter and part of the 5' UTR of the newly identified ycf9-psbM transciption unit, fused to the petD 5' UTR upstream of the FUD6 deletion. Accumulation of the ycf9-psbM dicistronic transcript was disrupted in the revertants, but monocistronic psbM mRNA accumulated normally. The FUD6 revertants demonstrate the ability of the Chlamydomonas chloroplast genome to undergo a large inversion without a deleterious effect on chloroplast function, reminiscent of events that have led to the evolutionary divergence of chloroplast genomes.

Animals↗
PubMed2001

State transitions reveal the dynamics and flexibility of the photosynthetic apparatus.

The chloroplast-based photosynthetic apparatus of plants and algae associates various redox cofactors and pigments with approximately 70 polypeptides to form five major transmembrane protein complexes. Among these are two photosystems that have distinct light absorption properties but work in series to produce reducing equivalents aimed at the fixation of atmospheric carbon. A short term chromatic adaptation known as 'State transitions' was discovered thirty years ago that allows photosynthetic organisms to adapt to changes in light quality and intensity which would otherwise compromise the efficiency of photosynthetic energy conversion. A two-decade research effort has finally unraveled the major aspects of the molecular mechanism responsible for State transitions, and their physiological significance has been revisited. This review describes how a-still elusive-regulatory kinase senses the physiological state of the photosynthetic cell and triggers an extensive supramolecular reorganization of the photosynthetic membranes. The resulting picture of the photosynthetic apparatus is that of a highly flexible energy convertor that adapts to the ever-changing intracellular demand for ATP and/or reducing power.

Cytochrome b Group↗
PubMed1995

Thermophilic bacilli have split cytochrome b genes for cytochrome b6 and subunit IV. First cloning of cytochrome b from a gram-positive bacterium (Bacillus stearothermophilus).

The genes of Bacillus stearothermophilus K1041 encoding cytochrome b(6) (Bacillus cytochrome b is referred to as cytochrome b(6) for its resemblance to plastid b6) and subunit IV of the quinol:cytochrome c oxidoreductase (bc1 complex) were cloned and sequenced. For preparation of the probe for cloning, polymerase chain reaction was carried out using oligonucleotide mixtures targeting for N-terminal regions of cytochrome bc and subunit IV of the thermophilic Bacillus PS3. The deduced amino acid sequences contained 224 residues of 25,425 daltons for cytochrome b(6) and 173 residues of 19,371 daltons for subunit IV, and both open reading frames were separated by 67 base pairs. Cytochrome b and subunit IV contained 4 and 3 hydrophobic transmembrane segments, respectively, indicating that the fourth segment of subunit IV (eighth segment of cytochrome b) is lacking. Four histidine residues supposed to ligand two protohemes were conserved, but the two His in the fourth segment were separated by 14 amino acid residues like cytochrome b6, not like mitochondrial cytochrome b. The residues that might have conferred the two quinol-binding sites were mostly conserved, but especially the third His residue in the fourth segment of mitochondrial cytochrome b was replaced by Arg in Bacillus cytochrome b6 as in cytochrome b6. These characteristics and quantitative comparison of the protein sequences indicate that this Bacillus sequence is unique and meanwhile rather close to the cyanobacteria-plastids type than the purple bacteria-mitochondria type.

Amino Acid Sequence↗
PubMed2005

A spontaneous tRNA suppressor of a mutation in the Chlamydomonas reinhardtii nuclear MCD1 gene required for stability of the chloroplast petD mRNA.

Numerous nuclear gene products are required for the correct expression of organellar genes. One such gene in the green alga Chlamydomonas reinhardtii is MCD1, whose product is required for stability of the chloroplast-encoded petD mRNA. In mcd1 mutants, which are non-photosynthetic, petD mRNA is degraded by a 5'-3' exonuclease activity, resulting in a failure to synthesize its product, subunit IV of the cytochrome b (6)/f complex. Here, we report the sequence of the wild-type MCD1 gene, which encodes a large and novel putative protein. Analysis of three mutant alleles showed that two harbored large deletions, but that one allele, mcd1-2, had a single base change resulting in a nonsense codon near the N-terminus. This same mutant allele can be suppressed by a second-site mutation in the nuclear MCD2 gene, whereas mcd2-1 cannot suppress the deletion in mcd1-1 (Esposito,D. Higgs,D.C. Drager,R.G. Stern, D.B. and Girard-Bascou,J. (2001) Curr. Genet., 39, 40-48). We report the cloning of mcd2-1, and show that the mutation lies in a tRNA(Ser)(CGA), which has been modified to translate the nonsense codon in mcd1-2. We discuss how the existence of a large tRNA(Ser) gene family may permit this suppression without pleiotropic consequences.

Algal Proteins↗
PubMed2002

Single point mutation in the Rieske iron-sulfur subunit of cytochrome b6/f leads to an altered pH dependence of plastoquinol oxidation in Arabidopsis.

The pgr1 mutant of Arabidopsis thaliana carries a single point mutation (P194L) in the Rieske subunit of the cytochrome b6/f (cyt b6/f) complex and is characterised by a reduced electron transport activity at saturating light intensities in vivo. We have investigated the electron transport in this mutant under in vitro conditions. Measurements of P700 reduction kinetics and of photosynthetic electron transport rates indicated that electron transfer from cyt b6/f to photosystem I is not generally reduced in the mutant, but that the pH dependence of this reaction is altered. The data imply that the pH-dependent inactivation of electron transport through cyt b6/f is shifted by about 1 pH unit to more alkaline pH values in pgr1 thylakoids in comparison with wild-type thylakoids. This interpretation was confirmed by determination of the transmembrane deltapH at different stromal pH values showing that the lumen pH in pgr1 mutant plants cannot drop below pH 6 reflecting most likely a shift of the pK and/or the redox potential of the oxidised Rieske protein.

Arabidopsis↗
PubMed2001

A nucleus-encoded suppressor defines a new factor which can promote petD mRNA stability in the chloroplast of Chlamydomonas reinhardtii.

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

Animals↗
PubMed1998

Spinach CSP41, an mRNA-binding protein and ribonuclease, is homologous to nucleotide-sugar epimerases and hydroxysteroid dehydrogenases.

Spinach CSP41 is part of a protein complex that binds to the 3' untranslated region (UTR) of petD precursor-mRNA, a chloroplast gene encoding subunit IV of the cytochrome b6/f complex. CSP41 cleaves the 3'-UTR of petD mRNA within the stem-loop structure, suggesting a key role in the control of chloroplast mRNA stability. We discovered that CSP41 is homologous to nucleotide-sugar epimerases and hydroxysteroid dehydrogenases while seeking distant homologs of these enzymes with a hidden Markov model-based search of Genpept. This analysis identified Synechocystis ORF, Accession 1652543 as a homolog. Subsequent analyses show that spinach CSP41 and Arabidopsis thaliana 2765081 are homologous to the Synechocystis ORF. Information from the solved 3D structures of epimerases and dehydrogenases and our motif analysis of these enzymes is used to predict domains on CSP41 that are important in binding and metabolism of mRNA. Cyanobacteria are among the earliest life forms, indicating that the divergence from a common ancestor of nucleotide-sugar epimerases and an mRNA binding protein with ribonuclease activity was ancient.

Amino Acid Sequence↗
PubMed1992

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex.

The gene encoding subunit IV of the cytochrome b6/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostoc sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella prototecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3'-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.

Amino Acid Sequence↗
PubMed2004

PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis.

Plastid gene expression is regulated by a variety of nuclear genes. We have isolated Arabidopsis thaliana proton gradient regulation 3 (pgr3) mutants, which display aberrant chlorophyll fluorescence because of defects in chloroplast gene expression. High chlorophyll fluorescence (HCF) because of a reduced level of the cytochrome b6/f complex was observed in two alleles, pgr3-1 and pgr3-2 but not in pgr3-3. In contrast, a transient increase in fluorescence after turning off the actinic light, which was ascribed to chloroplast NADPH dehydrogenase (NDH) activity, was impaired in pgr3-1 and pgr3-3 but not in pgr3-2. Both phenotypes were complemented by the introduction of a single gene, PGR3, encoding a protein containing 27 pentatrico-peptide repeat (PPR) motifs. PPR motifs are present in proteins functioning in the post-transcriptional regulation of organellar gene expression. The conserved threonine in the motif was substituted by isoleucine in the 15th and 12th PPR motifs in pgr3-1 and pgr3-2, respectively, and the conserved leucine by phenylalanine in the final incomplete motif of pgr3-3. We consider that the different domains of the PPR repeats in PGR3 might have different functions in conferring RNA stability and probably allowing translation as well as recognizing at least two distinct target RNAs.

Amino Acid Motifs↗