Kazuhito Inoue

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.

The major carotenoid in all known species of heliobacteria is the C30 carotenoid 4,4'-diaponeurosporene, not neurosporene.

Abstract The carotenoids of five species of heliobacteria (Heliobacillus mobilis, Heliophilum fasciatum, Heliobacterium chlorum, Heliobacterium modesticaldum, and Heliobacterium gestii) were examined by spectroscopic methods, and the C30 carotene 4,4′-diaponeurosporene was found to be the dominant pigment; heliobacteria were previously thought to contain the C40 carotenoid neurosporene. In addition, trace amounts of the C30 diapocarotenes diapolycopene, diapo-ζ-carotene, diapophytofluene, and diapophytoene were also found. Up to now, diapocarotenes have been found in only three species of chemoorganotrophic bacteria, but not in phototropic organisms. Furthermore, the esterifying alcohol of bacteriochlorophyll g from all known species of heliobacteria was determined to be farnesol (C15) instead of the usual phytol (C20). Heliobacteria may be unable to produce geranylgeranyol (C20).

Nitrogenase Fe protein-like Fe-S cluster is conserved in L-protein (BchL) of dark-operative protochlorophyllide reductase from Rhodobacter capsulatus

Dark‐operative protochlorophyllide reductase (DPOR) in bacteriochlorophyll biosynthesis is a nitrogenase‐like enzyme consisting of L‐protein (BchL‐dimer) as a reductase component and NB‐protein (BchN–BchB‐heterotetramer) as a catalytic component. Metallocenters of DPOR have not been identified. Here we report that L‐protein has an oxygen‐sensitive [4Fe–4S] cluster similar to nitrogenase Fe protein. Purified L‐protein from Rhodobacter capsulatus showed absorption spectra and an electron paramagnetic resonance signal indicative of a [4Fe–4S] cluster. The activity quickly disappeared upon exposure to air with a half‐life of 20 s. These results suggest that the electron transfer mechanism is conserved in nitrogenase Fe protein and DPOR L‐protein.

The nature of the primary electron acceptor in green sulfur bacteria

It was shown previously (Van de Meent, E.J., Kobayashi, M., Erkelens, C., Van Veelen, P.A., Amesz, J. and Watanabe, T. (1991) Biochim. Biophys. Acta 1058, 356–362) by means of HPLC, NMR and optical and mass spectroscopy that the primary electron acceptor of heliobacteria is 81-hydroxychlorophyll (Chl) a. In view of the spectral and functional similarities between this pigment and the primary electron acceptor of green sulfur bacteria, we have applied the same methods to various species of green sulfur bacteria (Prosthecochloris aestuarii, Chlorobium limicola, C. limicola f. thiosulfatophilum, C. vibrioforme and C. phaeovibrioides) in order to study the identity and the occurrence of the latter pigment. It was already shown from flash spectroscopic and reversed phase HPLC experiments on isolated membranes and solubilized membrane fractions of P. aestuarii that the most likely candidate for the primary acceptor is a pigment named bacteriochlorophyll (BChl) 663, which had been tentatively identified as a lipophilic from of BChl c. In this communication we will show by means of optical spectroscopy, 252Cf-plasma desorption mass spectroscopy and 1H-NMR that BChl 663 is an isomer of Chl a. This result again emphasizes the similarities between the reaction centers of green sulfur bacteria, heliobacteria and Photosystem I. By means of normal-phase HPLC analysis of the five species of green sulfur bacteria it is shown that BChl 663 is universally present and in comparable quantities in this group of photosynthetic bacteria. No other pigments with similar spectroscopic properties were detected.

NB-protein (BchN–BchB) of dark-operative protochlorophyllide reductase is the catalytic component containing oxygen-tolerant Fe–S clusters

Dark‐operative protochlorophyllide (Pchlide) oxidoreductase is a nitrogenase‐like enzyme consisting of the two components, L‐protein (BchL‐dimer) and NB‐protein (BchN–BchB‐heterotetramer). Here, we show that NB‐protein is the catalytic component with Fe–S clusters. NB‐protein purified from Rhodobacter capsulatus bound Pchlide that was readily converted to chlorophyllide a upon the addition of L‐protein and Mg‐ATP. The activity of NB‐protein was resistant to the exposure to air. A Pchlide‐free form of NB‐protein purified from a bchH‐lacking mutant showed an absorption spectrum suggesting the presence of Fe–S centers. Together with the Fe and sulfide contents, these findings suggested that NB‐protein carries two oxygen‐tolerant [4Fe–4S] clusters.

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.

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.

Light-independent isomerization of bacteriochlorophyll g to chlorophyll a catalyzed by weak acid in vitro

Abstract Rapid conversion of bacteriochlorophyll g (BChl g) to chlorophyll a (Chl a) was observed in acetone on addition of acid in the dark. The product, Chl a esterified with farnesol (Chl aF), was identified by liquid chromatography and fast atom bombardment mass spectrometry. Acid-catalyzed formation of 81-OH-Chl aF, a primary electron acceptor in the heliobacterial reaction center, was also observed in diethyl ether in the dark. These results suggest that acid-catalyzed isomerization is a candidate for the chemical evolution of BChl g to the more stable Chl a and that 81-OH-Chl aF can easily be synthesized from BChl g under weakly acidic conditions in the dark.

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.