Hidehiro Sakurai

The sites of electron donation of Photosystem I to methyl viologen

Abstract Effectiveness of methyl viologen as an electron acceptor was studied by oxygen polarography and millisecond time range flash photolysis spectroscopy at room temperature on three types of chloroplast preparation deficient in NADP+ photoreduction activity. HgCl2-treated chloroplasts which had completely lost Fe-S center B and also NADP+ photoreduction activity were still partially active in methyl viologen photoreduction. Compared with untreated chloroplasts, Vmax of the oxygen uptake in this preparation was almost halved and its apparent Km for methyl viologen was about 10 times greater. Photosystem I particles extracted with digitonin from the treated chloroplasts showed, in the absence of methyl viologen, a flash-induced absorption transient at 430 nm whose magnitude and decaying time were very similar to those of the particles extracted from untreated chloroplasts. However, the former required a concentration of methyl viologen for stabilization of P-700+ more than 10 times higher than control particles. The shape of the difference spectrum of the faster decaying component in the presence of methyl viologen was similar to P-430. Our conclusions are: (1) this spectral component represents the redox of Fe-S center A, and chloroplasts can transfer electrons from center X to center A even when their center B is destroyed; (2) center B is the main site of electron donation to methyl viologen, and center A can donate electrons to methyl viologen although with a lower affinity. Chloroplasts anaerobically photoinactivated under strongly reducing conditions in which electron transport between A0 and center X was impaired showed very low oxygen uptake activity which was almost insensitive to methyl viologen. Dependence of oxygen uptake on methyl viologen concentration by aerobically photoinactivated chloroplasts in which three Fe-S centers were partially destroyed somewhat resembled that of HgCl2-treated chloroplasts.

Promoting R & D in Photobiological Hydrogen Production Utilizing Mariculture-Raised Cyanobacteria

This review article explores the potential of using mariculture-raised cyanobacteria as solar energy converters of hydrogen (H2). The exploitation of the sea surface for large-scale renewable energy production and the reasons for selecting the economical, nitrogenase-based systems of cyanobacteria for H2 production, are described in terms of societal benefits. Reports of cyanobacterial photobiological H2 production are summarized with respect to specific activity, efficiency of solar energy conversion, and maximum H2 concentration attainable. The need for further improvements in biological parameters such as low-light saturation properties, sustainability of H2 production, and so forth, and the means to overcome these difficulties through the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering are also discussed. Finally, a possible mechanism for the development of economical large-scale mariculture operations in conjunction with international cooperation and social acceptance is outlined.

Ultraviolet absorption spectrum change induced by the interaction between chloroplast coupling factor 1 and ADP or ATP

Changes in the ultraviolet absorption spectrum took place when coupling factor 1 of spinach chloroplasts (CF1) was mixed with ATP or ADP. The difference spectrum had a maximum at about 278 nm and a minimum at about 250 nm. The profile of the difference spectrum inidcates a shift in the absorption spectrum of the ADP or ATP bound to CF1. The ratio of the maximal absorbance change at about 278 nm to the absorbance of CF1 at that wavelength was 0.027-0.048. The molar concentration of nucleotide required to give the maximal absorbance change was 2-3 times that of CF1 when Ca(2-) or Mg(2+) was present. AMP induced no significant spectral change. When ADP was added, the absorbance change reached a plateau within a few minutes if Mg(2-) or Ca(2+) was present. The absorbance change induced by ATP reached a plateau within a few minutes only when Ca(2+) was present. In the presence of Mg(2+), it reached a plateau of nearly the same level, but at a slower rate. The absorbance change was reduced in the presence of EDTA. PPi effectively inhibited the absorbance change induced by ADP and Mg(2+).

Function of the Reaction Center of Green Sulfur Bacteria

The reaction center (RC) of green sulfur bacteria belongs to the Fe‐S type RC, as do the photosystem I of oxygenic photosynthetic organisms and the RC of heliobacteria. The core parts of the green sulfur bacterial and the heliobacterial RC are assumed to be homodimeric, in contrast to those of purple bacteria, photosystem I and photosystem II. This paper describes recent advances in the study of the function of the green sulfur bacterial RC.

Inorganic sulfur oxidizing system in green sulfur bacteria

Green sulfur bacteria use various reduced sulfur compounds such as sulfide, elemental sulfur, and thiosulfate as electron donors for photoautotrophic growth. This article briefly summarizes what is known about the inorganic sulfur oxidizing systems of these bacteria with emphasis on the biochemical aspects. Enzymes that oxidize sulfide in green sulfur bacteria are membrane-bound sulfide-quinone oxidoreductase, periplasmic (sometimes membrane-bound) flavocytochrome c sulfide dehydrogenase, and monomeric flavocytochrome c (SoxF). Some green sulfur bacteria oxidize thiosulfate by the multienzyme system called either the TOMES (thiosulfate oxidizing multi-enzyme system) or Sox (sulfur oxidizing system) composed of the three periplasmic proteins: SoxB, SoxYZ, and SoxAXK with a soluble small molecule cytochrome c as the electron acceptor. The oxidation of sulfide and thiosulfate by these enzymes in vitro is assumed to yield two electrons and result in the transfer of a sulfur atom to persulfides, which are subsequently transformed to elemental sulfur. The elemental sulfur is temporarily stored in the form of globules attached to the extracellular surface of the outer membranes. The oxidation pathway of elemental sulfur to sulfate is currently unclear, although the participation of several proteins including those of the dissimilatory sulfite reductase system etc. is suggested from comparative genomic analyses.

Purification and characterization of ferredoxin-NAD(P)(+) reductase from the green sulfur bacterium Chlorobium tepidum.

Ferredoxin-NAD(P)(+) reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP(+) and NAD(+). When concentrations of NADP(+) exceeded 10 microM, NADP(+) photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD(+). It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.

Site-Directed Mutagenesis of the Anabaena sp. Strain PCC 7120 Nitrogenase Active Site To Increase Photobiological Hydrogen Production

ABSTRACT Cyanobacteria use sunlight and water to produce hydrogen gas (H2), which is potentially useful as a clean and renewable biofuel. Photobiological H2 arises primarily as an inevitable by-product of N2 fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H2 production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N2. Most of the 49 variants examined were deficient in N2-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N2 atmosphere significantly increased their in vivo rates of H2 production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H2 compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H2 in an aerobic, nitrogen-containing environment.

SoxAX Binding Protein, a Novel Component of the Thiosulfate-Oxidizing Multienzyme System in the Green Sulfur Bacterium Chlorobium tepidum

From the photosynthetic green sulfur bacterium Chlorobium tepidum (pro synon. Chlorobaculum tepidum), we have purified three factors indispensable for the thiosulfate-dependent reduction of the small, monoheme cytochrome c(554). These are homologues of sulfur-oxidizing (Sox) system factors found in various thiosulfate-oxidizing bacteria. The first factor is SoxYZ that serves as the acceptor for the reaction intermediates. The second factor is monomeric SoxB that is proposed to catalyze the hydrolytic cleavage of sulfate from the SoxYZ-bound oxidized product of thiosulfate. The third factor is the trimeric cytochrome c(551), composed of the monoheme cytochrome SoxA, the monoheme cytochrome SoxX, and the product of the hypothetical open reading frame CT1020. The last three components were expressed separately in Escherichia coli cells and purified to homogeneity. In the presence of the other two Sox factors, the recombinant SoxA and SoxX showed a low but discernible thiosulfate-dependent cytochrome c(554) reduction activity. The further addition of the recombinant CT1020 protein greatly increased the activity, and the total activity was as high as that of the native SoxAX-CT1020 protein complex. The recombinant CT1020 protein participated in the formation of a tight complex with SoxA and SoxX and will be referred to as SAXB (SoxAX binding protein). Homologues of the SAXB gene are found in many strains, comprising roughly about one-third of the thiosulfate-oxidizing bacteria whose sox gene cluster sequences have been deposited so far and ranging over the Chlorobiaciae, Chromatiaceae, Hydrogenophilaceae, Oceanospirillaceae, etc. Each of the deduced SoxA and SoxX proteins of these bacteria constitute groups that are distinct from those found in bacteria that apparently lack SAXB gene homologues.

Photobiological hydrogen production and nitrogenase activity in some heterocystous cyanobacteria

Publisher Summary This chapter discusses the activities of nitrogenase and H2 evolution in three cyanobacterial strains and some factors that affected these activities. Nitrogen-fixing heterocystous cyanobacteria are potential candidates for the development of photobiological hydrogen production systems. They produce H2 under aerobic conditions using water as an electron donor. For example A. variabilis IAM M-58 was most active in H2 production, and the amount of H2 produced was markedly higher than that of the other species. Hydrogen metabolism in these cyanobacteria involves at least three enzymes: nitrogenase, uptake hydrogenase, and bidirectional hydrogenase. Some researchers favor hydrogenase over nitrogenase as the hydrogen evolving system because of its high energy efficiency, but it was reported that continued production of H2 in air was mediated by nitrogenase in the heterocysts. To develop the hydrogen producing systems by cyanobacteria based on nitrogenase activity, it is important to find cyanobacterial strains which have a high activity of H2 production.

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl2 selectively destroys Fe−S center B (FB). The function of electron acceptors in FB-less PS I particles was studied by following the decay kinetics of P700+ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t1/2∼2.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t1/2∼0.25 ms). In FB-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in FB-less particles, electron transfer can proceed normally at room temperature from FX to FA and that the charge recombination between P700+ and FX-/A1- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of FB. Even in the presence of 60% glycerol, FB-less particles can transfer electrons to FA at room temperature as efficiently as untreated particles.