APX2 Is an Ascorbate Peroxidase-Related Protein that Regulates the Levels of Plastocyanin in Chlamydomonas.

The function of ascorbate peroxidase-related (APX-R) proteins, present in all green photosynthetic eukaryotes, remains unclear. This study focuses on APX-R from Chlamydomonas reinhardtii, namely, ascorbate peroxidase 2 (APX2). We showed that apx2 mutants exhibited a faster oxidation of the photosystem I primary electron donor, P700, upon sudden light increase and a slower re-reduction rate compared to the wild type, pointing to a limitation of plastocyanin. Spectroscopic, proteomic and immunoblot analyses confirmed that the phenotype was a result of lower levels of plastocyanin in the apx2 mutants. The redox state of P700 did not differ between wild type and apx2 mutants when the loss of function in plastocyanin was nutritionally complemented by growing apx2 mutants under copper deficiency. In this case, cytochrome c6 functionally replaces plastocyanin, confirming that lower levels of plastocyanin were the primary defect caused by the absence of APX2. Overall, the results presented here shed light on an unexpected regulation of plastocyanin level under copper-replete conditions, induced by APX2 in Chlamydomonas.

Alanine to serine substitutions drive thermal adaptation in a psychrophilic diatom cytochrome c6.

In this study, we investigate the thermodynamic mechanisms by which electron transfer proteins adapt to environmental temperature by directly comparing the redox properties and folding stability of a psychrophilic cytochrome c and a mesophilic homolog. Our model system consists of two cytochrome c6 proteins from diatoms: one adapted specifically to polar environments, the other adapted generally to surface ocean environments. Direct electrochemistry shows that the midpoint potential for the mesophilic homolog is slightly higher at all temperatures measured. Cytochrome c6 from the psychrophilic diatom unfolds with a melting temperature 10.4 °C lower than the homologous mesophilic cytochrome c6. Changes in free energy upon unfolding are identical, within error, for the psychrophilic and mesophilic protein; however, the chemical unfolding transition of the psychrophilic cytochrome c6 is more cooperative than for the mesophilic cytochrome c6. Substituting alanine residues found in the mesophile with serine found in corresponding positions of the psychrophile demonstrates that burial of the polar serine both decreases the thermal stability and decreases the midpoint potential. The mutagenesis data, combined with differences in the m-value of chemical denaturation, suggest that differences in solvent accessibility of the hydrophobic core underlie the adaptation of cytochrome c6 to differing environmental temperature.

Insights into the binding behavior of native and non-native cytochromes to photosystem I from Thermosynechococcus elongatus.

The binding of photosystem I (PS I) from Thermosynechococcus elongatus to the native cytochrome (cyt) c6 and cyt c from horse heart (cyt cHH) was analyzed by oxygen consumption measurements, isothermal titration calorimetry (ITC), and rigid body docking combined with electrostatic computations of binding energies. Although PS I has a higher affinity for cyt cHH than for cyt c6, the influence of ionic strength and pH on binding is different in the two cases. ITC and theoretical computations revealed the existence of unspecific binding sites for cyt cHH besides one specific binding site close to P700 Binding to PS I was found to be the same for reduced and oxidized cyt cHH Based on this information, suitable conditions for cocrystallization of cyt cHH with PS I were found, resulting in crystals with a PS I:cyt cHH ratio of 1:1. A crystal structure at 3.4-Å resolution was obtained, but cyt cHH cannot be identified in the electron density map because of unspecific binding sites and/or high flexibility at the specific binding site. Modeling the binding of cyt c6 to PS I revealed a specific binding site where the distance and orientation of cyt c6 relative to P700 are comparable with cyt c2 from purple bacteria relative to P870 This work provides new insights into the binding modes of different cytochromes to PS I, thus facilitating steps toward solving the PS I-cyt c costructure and a more detailed understanding of natural electron transport processes.

Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer.

A kinetic-LED-array-spectrophotometer (Klas) was recently developed for measuring in vivo redox changes of P700, plastocyanin (PCy), and ferredoxin (Fd) in the near-infrared (NIR). This spectrophotometer is used in the present work for in vitro light-induced measurements with various combinations of photosystem I (PSI) from tobacco and two different cyanobacteria, spinach plastocyanin, cyanobacterial cytochrome c6 (cyt. c6), and Fd. It is shown that cyt. c6 oxidation contributes to the NIR absorption changes. The reduction of (FAFB), the terminal electron acceptor of PSI, was also observed and the shape of the (FAFB) NIR difference spectrum is similar to that of Fd. The NIR difference spectra of the electron-transfer cofactors were compared between different organisms and to those previously measured in vivo, whereas the relative absorption coefficients of all cofactors were determined by using single PSI turnover conditions. Thus, the (840 nm minus 965 nm) extinction coefficients of the light-induced species (oxidized minus reduced for PC and cyt. c6, reduced minus oxidized for (FAFB), and Fd) were determined with values of 0.207 ± 0.004, - 0.033 ± 0.006, - 0.036 ± 0.008, and - 0.021 ± 0.005 for PCy, cyt. c6, (FAFB) (single reduction), and Fd, respectively, by taking a reference value of + 1 for P700+. The fact that the NIR P700 coefficient is larger than that of PCy and much larger than that of other contributing species, combined with the observed variability in the NIR P700 spectral shape, emphasizes that deconvolution of NIR signals into different components requires a very precise determination of the P700 spectrum.

Introgression of UfCyt c6, a thylakoid lumen protein from a green seaweed Ulva fasciata Delile enhanced photosynthesis and growth in tobacco.

Cytochromes are important components of photosynthetic electron transport chain. Here we report on genetic transformation of Cytochrome c6 (UfCyt c6) gene from Ulva fasciata Delile in tobacco for enhanced photosynthesis and growth. UfCyt c6 cDNA had an open reading frame of 330 bp encoding a polypeptide of 109 amino acids with a predicted molecular mass of 11.65 kDa and an isoelectric point of 5.21. UfCyt c6 gene along with a tobacco petE transit peptide sequence under control of CaMV35S promoter was transformed in tobacco through Agrobacterium mediated genetic transformation. Transgenic tobacco grew normal and exhibited enhanced growth as compared to wild type (WT) and vector control (VC) tobacco. Transgenic tobacco had higher contents of photosynthetic pigments and better ratios of photosynthetic pigments. The tobacco expressing UfCyt c6 gene exhibited higher photosynthetic rate and improved water use efficiency. Further activity of the water-splitting complex, photosystem II quantum yield, photochemical quenching, electron transfer rate, and photosynthetic yield were found comparatively higher in transgenic tobacco as compared to WT and VC tobacco. Alternatively basal quantum yield of non-photochemical processes in PSII and non-photochemical quenching were estimated lower in tobacco expressing UfCyt c6 gene. As a result of improved photosynthetic performance the transgenic tobacco had higher contents of sugar and starch, and exhibited comparatively better growth. To the best of our knowledge this is the first report on expression of UfCyt c6 gene from U. fasciata for improved photosynthesis and growth in tobacco.

Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

Interaction of photosystem I from Phaeodactylum tricornutum with plastocyanins as compared with its native cytochrome c6: Reunion with a lost donor.

In the Phaeodactylum tricornutum alga, as in most diatoms, cytochrome c6 is the only electron donor to photosystem I, and thus they lack plastocyanin as an alternative electron carrier. We have investigated, by using laser-flash absorption spectroscopy, the electron transfer to Phaeodactylum photosystem I from plastocyanins from cyanobacteria, green algae and plants, as compared with its own cytochrome c6. Diatom photosystem I is able to effectively react with eukaryotic acidic plastocyanins, although with less efficiency than with Phaeodactylum cytochrome c6. This efficiency, however, increases in some green alga plastocyanin mutants mimicking the electrostatics of the interaction site on the diatom cytochrome. In addition, the structure of the transient electron transfer complex between cytochrome c6 and photosystem I from Phaeodactylum has been analyzed by computational docking and compared to that of green lineage and mixed systems. Taking together, the results explain why the Phaeodactylum system shows a lower efficiency than the green systems, both in the formation of the properly arranged [cytochrome c6-photosystem I] complex and in the electron transfer itself.

Cytochrome c biogenesis in Campylobacter jejuni requires cytochrome c6 (CccA; Cj1153) to maintain apocytochrome cysteine thiols in a reduced state for haem attachment.

The microaerophilic food-borne pathogen Campylobacter jejuni uses complex cytochrome-rich respiratory chains for growth and host colonisation. Cytochrome c biogenesis requires haem ligation to reduced apocytochrome cysteines, catalysed by the cytochrome c synthase, CcsBA. While ccsBA could not be deleted, we showed that the thiol reductase DsbD and the CcsX homologue Cj1207 are involved in, but not essential for, cytochromes c biogenesis. Mutant phenotypic analyses and biochemical studies with purified proteins revealed that the mono-haem c-type cytochromes Cj1153 (CccA) and Cj1020 (CccB) and the di-haem Cj0037 (CccC) are electron donors to the cb-oxidase (CcoNOQP), with CccC being more efficient than CccA. Remarkably, cccA deletion or site-directed mutagenesis resulted in an almost complete loss of all other c-type cytochromes. Cytochrome c structural and biogenesis genes were still transcribed in the cccA deletion mutant and the quinol oxidase genes (cioAB) were up-regulated. Cytochrome c production could be rescued in this mutant by growth with exogenous dithiothreitol or L-cysteine, suggesting that in the absence of CccA, apocytochrome c haem binding motifs become oxidised, preventing haem attachment. Our results identify CccA, the most abundant periplasmic c-type cytochrome in C. jejuni, as a novel and unexpected protein required for cytochrome c biogenesis in this pathogen.

Quantum yield measurements of light-induced H2 generation in a photosystem I-[FeFe]-H2ase nanoconstruct.

The quantum yield for light-induced H2 generation was measured for a previously optimized bio-hybrid cytochrome c 6-crosslinked PSI(C13G)-1,8-octanedithiol-[FeFe]-H2ase(C97G) (PSI-H2ase) nanoconstruct. The theoretical quantum yield for the PSI-H2ase nanoconstruct is 0.50 molecules of H2 per photon absorbed, which equates to a requirement of two photons per H2 generated. Illumination of the PSI-H2ase nanoconstruct with visible light between 400 and 700 nm resulted in an average quantum yield of 0.10-0.15 molecules of H2 per photon absorbed, which equates to a requirement of 6.7-10 photons per H2 generated. A possible reason for the difference between the theoretical and experimental quantum yield is the occurrence of non-productive PSI(C13G)-1,8-octanedithiol-PSIC13G (PSI-PSI) conjugates, which would absorb light without generating H2. Assuming the thiol-Fe coupling is equally efficient at producing PSI-PSI conjugates as well as in producing PSI-H2ase nanoconstructs, the theoretical quantum yield would decrease to 0.167 molecules of H2 per photon absorbed, which equates to 6 photons per H2 generated. This value is close to the range of measured values in the current study. A strategy that purifies the PSI-H2ase nanoconstructs from the unproductive PSI-PSI conjugates or that incorporates different chemistries on the PSI and [FeFe]-H2ase enzyme sites could potentially allow the PSI-H2ase nanoconstruct to approach the expected theoretical quantum yield for light-induced H2 generation.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

Light-mediated hydrogen generation in Photosystem I: attachment of a naphthoquinone-molecular wire-Pt nanoparticle to the A1A and A1B sites.

The molecular wire-appended naphthoquinone 1-[15-(3-methyl-1,4-naphthoquinone-2-yl)]pentadecyl disulfide [(NQ(CH2)15S)2] has been incorporated into the A1A and A1B sites of Photosystem I (PS I) in the menB variant of Synechocystis sp. PCC 6803. Transient electron paramagnetic resonance studies show that the naphthoquinone headgroup displaces plastoquinone-9 from the A1A (and likely A1B) sites to a large extent. When a Pt nanoparticle is attached to the molecular wire by reductive cleavage of the disulfide and reaction with the resulting thiol, the PS I-NQ(CH2)15S-Pt nanoconstruct evolves dihydrogen at a rate of 67.3 µmol of H2 (mg of Chl)(-1) h(-1) [3.4 e(-) (PS I)(-1) s(-1)] after illumination for 1 h at pH 6.4. No dihydrogen is detected if wild-type PS I, which does not incorporate the quinone, is used or if either (NQ(CH2)15S)2 or the Pt nanoparticle is absent. Time-resolved optical studies of the PS I-NQ(CH2)15S-Pt nanoconstruct show that the lifetimes of the forward electron transfer to and reverse electron transfer from the iron-sulfur clusters are the same as in native PS I. Thus, electrons are not shuttled directly from the quinone to the Pt nanoparticle during either forward or reverse electron transfer. It is found that the rate of dihydrogen evolution in the PS I-NQ(CH2)15S-Pt nanoconstruct depends strongly on the concentration the sacrificial electron donor cytochrome c6. These observations can be explained if the iron-sulfur clusters are involved in stabilizing the electron; the ~50 ms residence time of the electron on FA or FB is sufficiently long to allow cytochrome c6 to reduce P700(+), thereby eliminating the recombination channel. In the absence of P700(+), slow electron transfer through the molecular wire to the Pt catalyst can occur, and hence, H2 evolution is observed.

Two types of fucoxanthin-chlorophyll-binding proteins I tightly bound to the photosystem I core complex in marine centric diatoms.

Intact fucoxanthin (Fucox)-chlorophyll (Chl)-binding protein I-photosystem I supercomplexes (FCPI-PSIs) were prepared by a newly developed simple fast procedure from centric diatoms Chaetoceros gracilis and Thalassiosira pseudonana to study the mechanism of their efficient solar energy accumulation. FCPI-PSI purified from C. gracilis contained 252 Chl a, 23 Chl c, 56 Fucox, 34 diadinoxanthin+diatoxanthin, 1 violaxanthin, 21 ß-carotene, and 2 menaquinone-4 per P700. The complex showed a high electron transfer activity at 185,000µmolmg Chl a(-1)·h(-1) to reduce methyl viologen from added cytochrome c6. We identified 14 and 21 FCP proteins in FCPI-PSI of C. gracilis and T. pseudonana, respectively, determined by N-terminal and internal amino acid sequences and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses. PsaO and a red lineage Chla/b-binding-like protein (RedCAP), Thaps3:270215, were also identified. Severe detergent treatment of FCPI-PSI released FCPI-1 first, leaving the FCPI-2-PSI-core complex. FCPI-1 contained more Chl c and showed Chl a fluorescence at a shorter wavelength than FCPI-2, suggesting an excitation-energy transfer from FCPI-1 to FCPI-2 and then to the PSI core. Fluorescence emission spectra at 17K in FCPI-2 varied depending on the excitation wavelength, suggesting two independent energy transfer routes. We formulated a model of FCPI-PSI based on the biochemical assay results.

Insights into the relationship between the haem-binding pocket and the redox potential of c6 cytochromes: four atomic resolution structures of c6 and c6-like proteins from Synechococcus sp. PCC 7002.

The structure of cytochrome c6C from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.03 Šresolution. This is the first structural report on the recently discovered cyanobacterial cytochrome c6-like proteins found in marine and nitrogen-fixing cyanobacteria. Despite high similarity in the overall three-dimensional fold between cytochromes c6 and c6C, the latter shows saliently different electrostatic properties in terms of surface charge distribution and dipole moments. Its midpoint redox potential is less than half of the value for typical c6 cytochromes and results mainly from the substitution of one residue in the haem pocket. Here, high-resolution crystal structures of mutants of both cytochromes c6 and c6C are presented, and the impact of the mutation of specific residues in the haem-binding pocket on the redox potential is discussed. These findings contribute to the elucidation of the structure-function relationship of c6-like cytochromes.

Photosystem I reduction in diatoms: as complex as the green lineage systems but less efficient.

Diatoms occupy a key branch in the evolutionary tree of oxygen-evolving photosynthetic organisms. Here, the electron transfer reaction mechanism from cytochrome c6 to photosystem I from the diatom Phaeodactylum tricornutum has been analyzed by laser-flash absorption spectroscopy. Kinetic traces of photosystem I reduction fit to biphasic curves, the analysis of the observed rate constants indicating that electron transfer occurs in a cytochrome c6/photosystem I transient complex, which undergoes a reorganization process from the initial encounter complex to the optimized final configuration. The mild ionic strength dependence of the rate constants makes evident the relatively weak electrostatically attractive nature of the interaction. Taken together, these results indicate that the "red" Phaeodactylum system is less efficient than "green" systems, both in the formation of the properly arranged (cytochrome c6/photosystem I) complex and in the electron transfer itself. The results obtained from cross-reactions with cytochrome c6 and photosystem I from cyanobacteria, green algae, and plants shed light on the different evolutionary pathway of the electron transfer to photosystem I in diatoms with regard to the way that it evolved in higher plants.

The role of a disulfide bridge in the stability and folding kinetics of Arabidopsis thaliana cytochrome c(6A).

Cytochrome c(6A) is a eukaryotic member of the Class I cytochrome c family possessing a high structural homology with photosynthetic cytochrome c(6) from cyanobacteria, but structurally and functionally distinct through the presence of a disulfide bond and a heme mid-point redox potential of +71mV (vs normal hydrogen electrode). The disulfide bond is part of a loop insertion peptide that forms a cap-like structure on top of the core α-helical fold. We have investigated the contribution of the disulfide bond to thermodynamic stability and (un)folding kinetics in cytochrome c(6A) from Arabidopsis thaliana by making comparison with a photosynthetic cytochrome c(6) from Phormidium laminosum and through a mutant in which the Cys residues have been replaced with Ser residues (C67/73S). We find that the disulfide bond makes a significant contribution to overall stability in both the ferric and ferrous heme states. Both cytochromes c(6A) and c(6) fold rapidly at neutral pH through an on-pathway intermediate. The unfolding rate for the C67/73S variant is significantly increased indicating that the formation of this region occurs late in the folding pathway. We conclude that the disulfide bridge in cytochrome c(6A) acts as a conformational restraint in both the folding intermediate and native state of the protein and that it likely serves a structural rather than a previously proposed catalytic role.

Residues PsaB Asp612 and PsaB Glu613 of photosystem I confer pH-dependent binding of plastocyanin and cytochrome c(6).

The binding and electron transfer between plastocyanin (pc) or cytochrome c(6) (cyt c(6)) and photosystem I (PSI) can be described by hydrophobic as well as electrostatic interactions. The two α helices, l and l' in PsaB and PsaA, respectively, are involved in forming the hydrophobic interaction site at the oxidizing site of PSI. To obtain mechanistic insights into the function of the two negatively charged residues D612 and E613, present in α helix l of PsaB, we exchanged both residues by site-directed mutagenesis with His and transformed a PsaB deficient mutant of Chlamydomonas reinhardtii. Flash-induced absorption spectroscopy revealed that PSI harboring the changes D612H and E613H had a high affinity toward binding of the electron donors and possessed an altered pH dependence of electron transfer with pc and cyt c(6). Despite optimized binding and electron transfer between the altered PSI and its electron donors, the mutant strain PsaB-D612H/E613H exhibited a strong light sensitive growth phenotype, indicating that decelerated turnover between pc/cyt c(6) and PSI with respect to electron transfer is deleterious to the cells.

CCS5, a thioredoxin-like protein involved in the assembly of plastid c-type cytochromes.

The c-type cytochromes are metalloproteins with a heme molecule covalently linked to the sulfhydryls of a CXXCH heme-binding site. In plastids, at least six assembly factors are required for heme attachment to the apo-forms of cytochrome f and cytochrome c(6) in the thylakoid lumen. CCS5, controlling plastid cytochrome c assembly, was identified through insertional mutagenesis in the unicellular green alga Chlamydomonas reinhardtii. The complementing gene encodes a protein with similarity to Arabidopsis thaliana HCF164, which is a thylakoid membrane-anchored protein with a lumen-facing thioredoxin-like domain. HCF164 is required for cytochrome b(6)f biogenesis, but its activity and site of action in the assembly process has so far remained undeciphered. We show that CCS5 is a component of a trans-thylakoid redox pathway and operates by reducing the CXXCH heme-binding site of apocytochrome c prior to the heme ligation reaction. The proposal is based on the following findings: 1) the ccs5 mutant is rescued by exogenous thiols; 2) CCS5 interacts with apocytochrome f and c(6) in a yeast two-hybrid assay; and 3) recombinant CCS5 is able to reduce a disulfide in the CXXCH heme-binding site of apocytochrome f.

Effect of crowding on the electron transfer process from plastocyanin and cytochrome c6 to photosystem I: a comparative study from cyanobacteria to green algae.

Plastocyanin and cytochrome c(6), the alternate donor proteins to photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I vary accordingly for cyanobacteria, algae and plants. The effect of different crowding agents on the kinetics of the reaction between plastocyanin or cytochrome c(6) and photosystem I from three different cyanobacteria, Synechocystis PCC 6803, Nostoc PCC 7119 and Arthrospira maxima, and a green alga, Monoraphidium braunii, has been investigated by laser flash photolysis, in order to elucidate how molecular crowding affects the interaction between the two donor proteins and photosystem I. The negative effect of viscosity on the interaction of the two donors with photosystem I for the three cyanobacterial systems is very similar, as studied by increasing sucrose concentration. Bovine serum albumin seems to alter the different systems in a specific way, probably by means of electrostatic interactions with the donor proteins. Ficoll and dextran behave in a parallel manner, favouring the interaction by an average factor of 2, although this effect is somewhat less pronounced in Nostoc. With regards to the eukaryotic system, a strong negative effect of viscosity is able to overcome the favourable effect of any crowding agent, maybe due to stronger donor/photosystem I electrostatic interactions or the structural nature of the eukaryotic photosystem I-enriched membrane particles.

Cytochrome c6-like protein as a putative donor of electrons to photosystem I in the cyanobacterium Nostoc sp. PCC 7119.

Most organisms performing oxygenic photosynthesis contain either cytochrome c(6) or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown. In this article, we describe a second cytochrome c(6) (the so called cytochrome c(6)-like protein), which is found in some cyanobacteria but is phylogenetically more related to plant cytochrome c(6A) than to cyanobacterial cytochrome c(6). In this article, we conclude that the cytochrome c(6)-like protein is a putative electron donor to photosystem I, but does play a role different to that of cytochrome c(6) and plastocyanin as it cannot accept electrons from cytochrome f. The existence of this third electron donor to PSI could explain why some cyanobacteria are able to grow photoautotrophically in the absence of both cytochrome c(6) and plastocyanin. In any way, the Cyt c(6)-like protein from Nostoc sp. PCC 7119 would be potentially utilized for the biohydrogen production, using cell-free photosystem I catalytic nanoparticles.

Structural and kinetic studies of imidazole binding to two members of the cytochrome c (6) family reveal an important role for a conserved heme pocket residue.

The amino acid at position 51 in the cytochrome c(6) family is responsible for modulating over 100 mV of heme midpoint redox potential. As part of the present work, the X-ray structure of the imidazole adduct of the photosynthetic cytochrome c(6) Q51V variant from Phormidium laminosum has been determined. The structure reveals the axial Met ligand is dissociated from the heme iron but remains inside the heme pocket and the Ω-loop housing the Met ligand is stabilized through polar interactions with the imidazole and heme propionate-6. The latter is possible owing to a 180° rotation of both heme propionates upon imidazole binding. From equilibrium and kinetic studies, a Val residue at position 51 increases the stability of the Fe-S(Met) interaction and also affects the dynamics associated with imidazole binding. In this respect, the k (obs) for imidazole binding to Arabidopsis thaliana cytochrome c(6A), which has a Val at the position equivalent to position 51 in photosynthetic cytochrome c(6), was found to be independent of imidazole concentration, indicating that the binding process is limited by the Met dissociation rate constant (about 1 s(-1)). For the cytochrome c(6) Q51V variant, imidazole binding was suppressed in comparison with the wild-type protein and the V52Q variant of cytochrome c(6A) was found to bind imidazole readily. We conclude that the residue type at position 51/52 in the cytochrome c(6) family is additionally responsible for tuning the stability of the heme iron-Met bond and the dynamic properties of the ferric protein fold associated with endogenous ligand binding.