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or plastocyanin, or both, to transfer electrons from cytochrome b(6)-f to photosystem I. Even though plastocyanin has superseded cytochrome c(6) along evolution, plants contain a modified cytochrome c(6), the so called cytochrome c(6A), whose function still remains unknown.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.

Purification of plastocyanin and cytochrome c6 from plants, green algae, and cyanobacteria.

Plastocyanin and cytochrome c6 are widely distributed over the oxygen-evolving photosynthetic organisms. The two proteins are functionally equivalent, but strongly differ in their global electrostatic charge. In fact, they are acidic in eukaryotes, but either neutral or basic in cyanobacteria. Such a difference in their electrostatic features is a critical factor in designing the purification procedure, which must thus be modified and adapted accordingly. This chapter reports the methods for producing (including cell cultures), isolating, and purifying plastocyanin and cytochrome c6--which greatly differ in their isoelectric point--from a number of eukaryotic and prokaryotic organisms.

Self-organized photosynthetic nanoparticle for cell-free hydrogen production.

There is considerable interest in making use of solar energy through photosynthesis to create alternative forms of fuel. Here, we show that photosystem I from a thermophilic bacterium and cytochrome-c(6) can, in combination with a platinum catalyst, generate a stable supply of hydrogen in vitro upon illumination. The self-organized platinization of the photosystem I nanoparticles allows electron transport from sodium ascorbate to photosystem I via cytochrome-c(6) and finally to the platinum catalyst, where hydrogen gas is formed. Our system produces hydrogen at temperatures up to 55 degrees C and is temporally stable for >85 days with no decrease in hydrogen yield when tested intermittently. The maximum yield is approximately 5.5 micromol H(2) h(-1) mg(-1) chlorophyll and is estimated to be approximately 25-fold greater than current biomass-to-fuel strategies. Future work will further improve this yield by increasing the kinetics of electron transfer, extending the spectral response and replacing the platinum catalyst with a renewable hydrogenase.

Purification and characterization of cytochrome c(6) from Acaryochloris marina.

Cytochrome c(6), (cyt c(6)) a soluble monoheme electron transport protein, was isolated and characterized from the chlorophyll d-containing cyanobacterium Acaryochoris marina, the type strain MBIC11017. The protein was purified using ammonium sulfate precipitation, ion exchange and gel filtration column chromatography, and fast performance liquid chromatography. Its molecular mass and pI have been determined to be 8.87 kDa and less than 4.2, respectively, by mass spectrometry and isoelectrofocusing (IEF). The protein has an alpha helical structure as indicated by CD (circular dichroism) spectroscopy and a reduction midpoint potential (E(m)) of +327 mV versus the normal hydrogen electrode (NHE) as determined by redox potentiometry. Its potential role in electron transfer processes is discussed.

Cloning, expression and purification of cytochrome c(6) from the brown alga Hizikia fusiformis and complete X-ray diffraction analysis of the structure.

The primary sequence of cytochrome c(6) from the brown alga Hizikia fusiformis has been determined by cDNA cloning and the crystal structure has been solved at 1.6 A resolution. The crystal belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 84.58, c = 232.91 A and six molecules per asymmetric unit. The genome code, amino-acid sequence and crystal structure of H. fusiformis cytochrome c(6) were most similar to those of red algal cytochrome c(6). These results support the hypothesis that brown algae acquired their chloroplasts via secondary endosymbiosis involving a red algal endosymbiont and a eukaryote host.

The novel cytochrome c6 of chloroplasts: a case of evolutionary bricolage?

Cytochrome c6 has long been known as a redox carrier of the thylakoid lumen of cyanobacteria and some eukaryotic algae that can substitute for plastocyanin in electron transfer. Until recently, it was widely accepted that land plants lack a cytochrome c6. However, a homologue of the protein has now been identified in several plant species together with an additional isoform in the green alga Chlamydomonas reinhardtii. This form of the protein, designated cytochrome c6A, differs from the 'conventional' cytochrome c6 in possessing a conserved insertion of 12 amino acids that includes two absolutely conserved cysteine residues. There are conflicting reports of whether cytochrome c6A can substitute for plastocyanin in photosynthetic electron transfer. The evidence for and against this is reviewed and the likely evolutionary history of cytochrome c6A is discussed. It is suggested that it has been converted from a primary role in electron transfer to one in regulation within the chloroplast, and is an example of evolutionary 'bricolage'.

Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.

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.

Purification of plastocyanin and cytochrome c6 from plants, green algae, and cyanobacteria.

Plastocyanin and cytochrome c6 are widely distributed over the oxygen-evolving photosynthetic organisms. The two proteins are functionally equivalent, but strongly differ in their global electrostatic charge. In fact, they are acidic in eukaryotes, but either neutral or basic in cyanobacteria. The difference in their electrostatic features is a critical factor in designing the purification procedure, which must be modified and adapted accordingly. This chapter reports the methods for producing (including cell cultures), isolating and purifying plastocyanin and cytochrome c6-which greatly differ in their isoelectric point-from a number of eukaryotic and prokaryotic organisms.