Hirofumi Nishihara

Structural basis for a [4Fe-3S] cluster in the oxygen-tolerant membrane-bound [NiFe]-hydrogenase

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H2-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H2 → 2H+ + 2e− (ref. 1). In contrast to the well-studied O2-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O2-tolerant H2 oxidation activity; however, the mechanism of O2 tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32 Å. We find that the proximal iron-sulphur (Fe-S) cluster of MBH has a [4Fe-3S] structure coordinated by six cysteine residues—in contrast to the [4Fe-4S] cubane structure coordinated by four cysteine residues found in the proximal Fe-S cluster of the standard enzymes—and that an amide nitrogen of the polypeptide backbone is deprotonated and additionally coordinates the cluster when chemically oxidized, thus stabilizing the superoxidized state of the cluster. The structure of MBH is very similar to that of the O2-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O2 tolerance of MBH is attributable to the unique proximal Fe-S cluster; we propose that the cluster is not only a component of the electron transfer for the catalytic cycle, but that it also donates two electrons and one proton crucial for the appropriate reduction of O2 in preventing the formation of an unready, inactive state of the enzyme.

Light-driven Hydrogen Production by a Hybrid Complex of a [NiFe]-Hydrogenase and the Cyanobacterial Photosystem I

Abstract In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a ‘hard-wired’ protein complex consisting of a hydrogenase and photosystem I (hydrogenase–PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the β-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase–PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase–PSI complex displayed light-driven hydrogen production at a rate of 0.58 μmol H2·mg chlorophyll−1·h−1. The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.

Hydrogenovibrio marinus gen. nov., sp. nov., a marine obligately chemolithoautotrophic hydrogen-oxidizing bacterium

The name Hydrogenovibrio marinus gen. nov., sp. nov. is proposed for an obligately chemolithoautotrophic, mesophilic, gram-negative, motile, comma-shaped, aerobic, hydrogen-oxidizing bacterium that was isolated from seawater. The optimum temperature and NaCl concentration for growth are 37°C and 0.5 M, respectively. The guanine-plus-cytosine content of the DNA is 44.1 mol%. The ubiquinone is ubiquinone-8, and the major cellular fatty acids are C16:0, C18:0, and C16:1 acids. The type strain of this species is strain MH-110 (= JCM 7688).

Occurrence of hydrogen-oxidizing Ralstonia species as primary microorganisms in the Mt. Pinatubo volcanic mudflow deposits

Abstract Ralstonia eutropha is an aerobic, hydrogen-oxidizing, facultative chemolithotrophic bacterium. Recently, R. eutropha-related bacteria have been found to predominate in the cultural bacterial communities of fresh mudflow deposits from Mt. Pinatubo, the Philippines. In the present study, the R. eutropha-related strains were examined for their chemolithotrophic growth in the presence of H2, O2, amd CO2 (85 :5 :10) and for their phylogenetic relationship with R. eutropha. Eleven of the 12 strains tested grew in the presence of H2, O2, and CO2, although the growth was not as rapid as that of the well-characterized H2-oxidizing strain, R. eutropha H16. To examine the hydrogenase activity, membrane and soluble fractions were prepared from R. eutropha-related strain 1245 and its activity was assayed with methylene blue and NAD as electron acceptors. A significant hydrogenase activity was detected in both membrane and soluble fractions of the cell-free extract. The 16S rDNA analysis of the 12 strains of R. eutropha-related bacteria revealed that they could be classified into two clusters, both of which were clearly separated from the cluster of R. eutropha sensu stricto. The probable ecological niche of H2-oxidizing Ralstonia species in the volcanic mudflow deposits was examined.

Different properties of gene products of three sets ribulose 1,5-bisphosphate carboxylase/oxygenase from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110

Abstract Hydrogenovibrio marinus strain MH-110 is an obligately lithoautotrophic hydrogen-oxidizing bacterium, possessing three sets of the genes for ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO); one form II type (L x ) and two form I type (L 8 S 8 ) enzymes. The genes for the form I type enzymes are named cbbLS-1 and cbbLS-2 , and that for the form II type enzyme is named cbbM . These three sets of genes were cloned in plasmid vectors, and the expression of the genes in E. coli cells were studied. The three RubisCOs were purified through the same steps, and their specificity factors (τ values) were measured. The τ values of CbbLS-1 and CbbLS-2 were higher than that of CbbM, but lower than those of other form I RubisCOs. The specific activity of CbbM was very low and CbbM was inactivated easily during the process of purification. Immunochemical analyses revealed that the antibody against form I type reacted only with CbbL-1 and CbbL-2, and the antibody against form II type reacted only with CbbM. The antibody to neither form of RubisCO demonstrated cross-reactivity for the other form.

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis

BackgroundConversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst.ResultsThe two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts.ConclusionsAsymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.

Purification and biochemical characterization of a membrane‐bound [NiFe]‐hydrogenase from a hydrogen‐oxidizing, lithotrophic bacterium, Hydrogenophaga sp. AH‐24

Membrane-bound [NiFe]-hydrogenase from Hydrogenophaga sp. AH-24 was purified to homogeneity. The molecular weight was estimated as 100+/-10 kDa, consisting of two different subunits (62 and 37 kDa). The optimal pH values for H(2) oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H(2) oxidation/H(2) evolution) was 1.61 x 10(2) at pH 7.0. The optimal temperature was 75 degrees C. The enzyme was quite stable under air atmosphere (the half-life of activity was c. 48 h at 4 degrees C), which should be important to function in the aerobic habitat of the strain. The enzyme showed high thermal stability under anaerobic conditions, which retained full activity for over 5 h at 50 degrees C. The activity increased up to 2.5-fold during incubation at 50 degrees C under H(2). Using methylene blue as an electron acceptor, the kinetic constants of the purified membrane-bound homogenase (MBH) were V(max)=336 U mg(-1), k(cat)=560 s(-1), and k(cat)/K(m)=2.24 x 10(7) M(-1) s(-1). The MBH exhibited prominent electron paramagnetic resonance signals originating from [3Fe-4S](+) and [4Fe-4S](+) clusters. On the other hand, signals originating from Ni of the active center were very weak, as observed in other oxygen-stable hydrogenases from aerobic H(2)-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga.

Thiosulfate oxidation by a moderately thermophilic hydrogen-oxidizing bacterium, Hydrogenophilus thermoluteolus

The moderately thermophilic Betaproteobacterium, Hydrogenophilus thermoluteolus, not only oxidizes hydrogen, the principal electron donor for growth, but also sulfur compounds including thiosulfate, a process enabled by sox genes. A periplasmic extract of H. thermoluteolus showed significant thiosulfate oxidation activity. Ten genes apparently involved in thiosulfate oxidation (soxEFCDYZAXBH) were found on a 9.7-kb DNA fragment of the H. thermoluteolus chromosome. The proteins SoxAX, which represent c-type cytochromes, were co-purified from the cells of H. thermoluteolus; they enhanced the thiosulfate oxidation activity of the periplasmic extract when added to the latter.

Structural basis of the redox switches in the NAD(+)-reducing soluble [NiFe]-hydrogenase

How a hydrogenase protects its active site Hydrogen-metabolizing organisms use an [NiFe]-hydrogenase to catalyze hydrogen oxidation. One type of [NiFe]-hydrogenase, the NAD+-reducing soluble [NiFe]-hydrogenase (SH), couples reduction of NAD+ to the oxidation of hydrogen. Shomura et al. solved the structure of SH from an H2-oxidizing bacterium in both the air-oxidized and the active reduced state. In the reduced state, the NiFe catalytic center in SH has the same ligand coordination as in other [NiFe]-hydrogenases. However, the air-oxidized active site has an unusual coordination geometry that would prevent O2 from accessing the site and so may protect against irreversible oxidation. Science, this issue p. 928 Coordination geometry at the active site may protect a hydrogenase enzyme from irreversible oxidation. NAD+ (oxidized form of NAD:nicotinamide adenine dinucleotide)–reducing soluble [NiFe]-hydrogenase (SH) is phylogenetically related to NADH (reduced form of NAD+):quinone oxidoreductase (complex I), but the geometrical arrangements of the subunits and Fe–S clusters are unclear. Here, we describe the crystal structures of SH in the oxidized and reduced states. The cluster arrangement is similar to that of complex I, but the subunits orientation is not, which supports the hypothesis that subunits evolved as prebuilt modules. The oxidized active site includes a six-coordinate Ni, which is unprecedented for hydrogenases, whose coordination geometry would prevent O2 from approaching. In the reduced state showing the normal active site structure without a physiological electron acceptor, the flavin mononucleotide cofactor is dissociated, which may be caused by the oxidation state change of nearby Fe–S clusters and may suppress production of reactive oxygen species.

High Thermal Stability and Unique Trimer Formation of Cytochrome c′ from Thermophilic Hydrogenophilus thermoluteolus

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c′ (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c′ (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c′ family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c′. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.