Brownian dynamics simulations of the interaction of Chlamydomonas cytochrome f with plastocyanin and cytochrome c6.
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The interaction of Chlamydomonas cytochrome f (cyt f) with either Chlamydomonas plastocyanin (PC) or Chlamydomonas cytochrome c(6) (cyt c(6)) was studied using Brownian dynamics simulations. The two electron acceptors (PC and cyt c(6)) were found to be essentially interchangeable despite a lack of sequence homology and different secondary structures (beta-sheet for PC and alpha-helix for cyt c(6)). Simulations using PC and cyt c(6) interacting with cyt f showed approximately equal numbers of successful complexes and calculated rates of electron transfer. Cyt f-PC and cyt f-cyt c(6) showed the same types of interactions. Hydrophobic residues surrounding the Y1 ligand to the heme on cyt f interacted with hydrophobic residues on PC (surrounding the H87 ligand to the Cu) or cyt c(6) (surrounding the heme). Both types of complexes were stabilized by electrostatic interactions between K65, K188, and K189 on cyt f and conserved anionic residues on PC (E43, D44, D53, and E85) or cyt c(6) (E2, E70, and E71). Mutations on cyt f had identical effects on its interaction with either PC or cyt c(6). K65A, K188A, and K189A showed the largest effects whereas residues such as K217A, R88A, and K110A, which are located far from the positive patch on cyt f, showed very little inhibition. The effect of mutations observed in Brownian dynamics simulations paralleled those observed in experiments. (+info)
Copper response element and Crr1-dependent Ni(2+)-responsive promoter for induced, reversible gene expression in Chlamydomonas reinhardtii.
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The Cpx1 and Cyc6 genes of Chlamydomonas reinhardtii are activated in copper-deficient cells via a signal transduction pathway that requires copper response elements (CuREs) and a copper response regulator defined by the CRR1 locus. The two genes can also be activated by provision of nickel or cobalt ions in the medium. The response to nickel ions requires at least one CuRE and also CRR1 function, suggesting that nickel interferes with a component in the nutritional copper signal transduction pathway. Nickel does not act by preventing copper uptake/utilization because (i) holoplastocyanin formation is unaffected in Ni(2+)-treated cells and (ii) provision of excess copper cannot reverse the Ni-dependent activation of the target genes. The CuRE is sufficient for conferring Ni-responsive expression to a reporter gene, which suggests that the system has practical application as a vehicle for inducible gene expression. The inducer can be removed either by replacing the medium or by chelating the inducer with excess EDTA, either of which treatments reverses the activation of the target genes. (+info)
Structure and function of photosystem I: interaction with its soluble electron carriers and external antenna systems.
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Photosystem I (PS I) is a large membrane protein complex that catalyzes the first step of solar conversion, the light-induced transmembrane electron transfer, and generates reductants for CO2 assimilation. It consists of 12 different proteins and 127 cofactors that perform light capturing and electron transfer. The function of PS I includes inter-protein electron transfer between PS I and smaller soluble electron transfer proteins. The structure of PS I is discussed with respect to the potential docking sites for the soluble electron acceptors, ferredoxin/flavodoxin, at the stromal side and the soluble electron donors, cytochrome c6/plastocyanin, at the luminal side of the PS I complex. Furthermore, the potential interaction sites with the peripheral antenna proteins are discussed. (+info)
The efficient functioning of photosynthesis and respiration in Synechocystis sp. PCC 6803 strictly requires the presence of either cytochrome c6 or plastocyanin.
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In cyanobacteria, cytochrome c6 and plastocyanin are able to replace each other as redox carriers in the photosynthetic and respiratory electron transport chains with the synthesis of one or another protein being regulated by the copper concentration in the culture medium. However, the presence of a third unidentified electron carrier has been suggested. To address this point, we have constructed two deletion mutants of the cyanobacterium Synechocystis sp. PCC 6803, each variant lacking either the petE or petJ gene, which respectively codes for the copper or heme protein. The photoautotrophic and heterotrophic growth rate of the two mutants in copper-free and copper-supplemented medium as well as their photosystem I reduction kinetics in vivo were compared with those of wild-type cells. The two mutant strains grow at equivalent rates and show similar in vivo photosystem I reduction kinetics as wild-type cells when cultured in media that allow the expression of just one of the two electron donor proteins, but their ability to grow and reduce photosystem I is much lower when neither cytochrome c6 nor plastocyanin is expressed. These findings indicate that the normal functioning of the cyanobacterial photosynthetic and respiratory chains obligatorily depends on the presence of either cytochrome c6 or plastocyanin. (+info)
The hydrophobic recognition site formed by residues PsaA-Trp651 and PsaB-Trp627 of photosystem I in Chlamydomonas reinhardtii confers distinct selectivity for binding of plastocyanin and cytochrome c6.
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On the lumenal side of photosystem I (PSI), each of the two large core subunits, PsaA and PsaB, expose a conserved tryptophan residue to the surface. PsaB-Trp(627) is part of the hydrophobic recognition site that is essential for tight binding of the two electron donors plastocyanin and cytochrome c(6) to the donor side of PSI (Sommer, F., Drepper, F., and Hippler, M. (2002) J. Biol. Chem. 277, 6573-6581). To examine the function of PsaA-Trp(651) in binding and electron transfer of both donors to PSI, we generated the mutants PsaA-W651F and PsaA-W651S by site-directed mutagenesis and biolistic transformation of Chlamydomonas reinhardtii. The protein-protein interaction and the electron transfer between the donors and PSI isolated from the mutants were analyzed by flash absorption spectroscopy. The mutation PsaA-W651F completely abolished the formation of a first order electron transfer complex between plastocyanin (pc) and the altered PSI and increased the dissociation constant for binding of cytochrome (cyt) c(6) by more than a factor of 10 as compared with wild type. Mutation of PsaA-Trp(651) to Ser had an even larger impact on the dissociation constant. The K(D) value increased another 2-fold when the values obtained for the interaction and electron transfer between cyt c(6) and PSI from PsaA-W651S and PsaA-W651F are compared. In contrast, binding and electron transfer of pc to PSI from PsaA-W651S improved as compared with PSI from PsaA-W651F and admitted the formation of an inter-molecular electron transfer complex, resulting in a K(D) value of about 554 microm that is still five times higher than observed for wild type. These results demonstrate that PsaA-Trp(651) is, such as PsaB-Trp(627), crucial for high affinity binding of pc and cyt c(6) to PSI. Our results also indicate that the highly conserved structural recognition motif that is formed by PsaA-Trp(651) and PsaB-Trp(627) confers a differential selectivity in binding of both donors to PSI. (+info)
Redox properties of Arabidopsis cytochrome c6 are independent of the loop extension specific to higher plants.
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Cytochrome c6 (cytc6) from Arabidopsis differs from the cyanobacterial and algal homologues in several redox properties. It is possible that these differences might be due to the presence of a 12 amino acid residue loop extension common to higher plant cytc6 proteins. However, homology modelling suggests this is not the case. We report experiments to test if differences in biochemical properties could be due to this extension. Analysis of mutant forms of Arabidopsis cytc6 in which the entire extension was lacking, or a pair of cysteine residues in the extension had been exchanged for serine, revealed no significant effect of these changes on either the redox potential of the haem group or the reactivity towards Photosystem I (PSI). We conclude that the differences in properties are due to more subtle unidentified differences in structure, and that the sequence extension in the higher plant proteins has a function yet to be identified. (+info)
Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.
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Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways. (+info)
NMR analysis of the transient complex between membrane photosystem I and soluble cytochrome c6.
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A structural analysis of the surface areas of cytochrome c(6), responsible for the transient interaction with photosystem I, was performed by NMR transverse relaxation-optimized spectroscopy. The hemeprotein was titrated by adding increasing amounts of the chlorophyllic photosystem, and the NMR spectra of the free and bound protein were analyzed in a comparative way. The NMR signals of cytochrome c(6) residues located at the hydrophobic and electrostatic patches, which both surround the heme cleft, were specifically modified by binding. The backbones of internal residues close to the hydrophobic patch of cytochrome c(6) were also affected, a fact that is ascribed to the conformational changes taking place inside the hemeprotein when interacting with photosystem I. To the best of our knowledge, this is the first structural analysis by NMR spectroscopy of a transient complex between soluble and membrane proteins. (+info)