Language：English  

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.

DOI： 10.1126/science.aan4497

Web of Science

PubMed

researchmap

Language：English  

Membrane-bound respiratory [NiFe]-hydrogenase (MBH), a H
₂-uptake enzyme found in the periplasmic space of bacteria, catalyses the oxidation of dihydrogen: H
₂ →2H
⁺ + 2e
^- (ref. 1). In contrast to the well-studied O
₂-sensitive [NiFe]-hydrogenases (referred to as the standard enzymes), MBH has an O
₂-tolerant H
₂ oxidation activity; however, the mechanism of O
₂ tolerance is unclear. Here we report the crystal structures of Hydrogenovibrio marinus MBH in three different redox conditions at resolutions between 1.18 and 1.32Å 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 O
₂-sensitive standard enzymes except for the proximal Fe-S cluster. Our results give a reasonable explanation why the O
₂ 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 O
₂ in preventing the formation of an unready, inactive state of the enzyme.

DOI： 10.1038/nature10504

researchmap

Publisher：Chemistry Letters  

Various types of artificial hydrogenase model complexes have been synthesized by imitating the active sites of natural [NiFe] hydrogenases. However, the control of their catalytic properties after synthesizing complexes by using modifiable groups is still difficult because predesigned modifiable groups can react easily with metal cations during multistep synthesis. In this study, we successfully synthesized a newly designed [NiRu] complex with modifiable OH groups, which could act as a homogeneous H<inf>2</inf> oxidation catalyst by using Cu2+ as an electron acceptor.

DOI： 10.1093/chemle/upaf057

Web of Science

Scopus

Language：Japanese   Publisher：The Society for Biotechnology, Japan  

DOI： 10.34565/seibutsukogaku.103.2_61

CiNii Research

Language：English   Publisher：Chemistry an Asian Journal  

The increasing levels of carbon dioxide (CO<inf>2</inf>) in the atmosphere, primarily due to the use of fossil fuels, pose a significant threat to the environment and necessitate urgent action to mitigate climate change. Carbon capture and utilization technologies that can convert CO<inf>2</inf> into economically valuable compounds have gained attention as potential solutions. Among these technologies, biocatalytic CO<inf>2</inf> hydrogenation using bacterial whole cells shows promise for the efficient conversion of CO<inf>2</inf> into formate, a valuable chemical compound. Although it was discovered nearly a century ago, comprehensive reviews focusing on the utilization of whole-cell bacteria as the biocatalyst in this area remain relatively limited. Therefore, this review provides an analysis of the progress, strategies, and key findings in this field. It covers the use of living cells, resting cells, or genetically modified bacteria as biocatalysts to convert CO<inf>2</inf> into formate, either naturally or with the integration of electrochemical and protochemical techniques as sources of protons and electrons. By consolidating the current knowledge in this field, this review article aims to serve as a valuable resource for researchers and practitioners interested in understanding the recent progress, challenges, and potential applications of bacterial whole cell catalyzed CO<inf>2</inf> hydrogenation into formate.

DOI： 10.1002/asia.202400468

Web of Science

Scopus

PubMed

Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

Replacing wasteful metal-based reducing agents with H<inf>2</inf> is an important goal for green chemistry. For this reason, we outline our design principles for building catalysts that use electrons from hydrogen to activate organohalides for reaction. These designs rely on an electron-withdrawing ligand to support low-valent metal centers, an electron-donating ligand to support oxidative addition, and the capacity for vacant sites to allow substrate docking. We begin by outlining our previous work in this field before describing a new rhodium complex that activates a particularly stubborn organohalide, 2,2-dibromopropane, using electrons from hydrogen. We then react this activated organohalide with styrene to generate a synthetically useful fragment of murraol in an efficient manner.

DOI： 10.1021/acs.organomet.4c00360

Web of Science

Scopus

researchmap

Publisher：ACS Sustainable Chemistry and Engineering  

The electrochemical reduction of nicotinamide adenine dinucleotide (NAD) using water as a hydrogen source is a promising strategy for the efficient and environmentally friendly production of the active enzymatic cofactor 1,4-NADH, which is a key for the further application of enzymatic systems in various industrial fields. However, the efficient regeneration of 1,4-NADH (NADH-reg) remains a major challenge. The rate-limiting step in the electrochemical conversion of the oxidized NAD is the second electron transfer to an NAD radical, which is formed by the reduction of NAD⁺, at a large overpotential, whereas the other side reactions proceed readily. To surmount this obstacle and promote NADH-reg, we used Ni nanoparticle-loaded TiO<inf>2</inf> on a Ti electrode (Ni-TOT) containing a sufficient number of defects as active sites, which are formed at 300 °C in the H<inf>2</inf> atmosphere. Ni-TOT facilitated the formation of enzymatically active 1,4-NADH with a superior yield and significantly reduced overpotential compared to those on untreated TOT. We found that hydrogen spillover promotes the formation of active sites on Ni-TOT. This study highlights the potential of engineered defect-enriched electrodes as a means to advance NADH-reg.

DOI： 10.1021/acssuschemeng.4c02313

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

C-phycocyanin (CPC), which contains open-chain tetrapyrroles, is a major light-harvesting red-fluorescent protein with an important role in aquatic photosynthesis. Recently, we reported a non-conventional CPC from Thermoleptolyngbya sp. O-77 (CPC<inf>O77</inf>) that contains two different structures, i.e., a hexameric structure and a non-conventional octameric structure. However, the assembly and disassembly mechanisms of the non-conventional octameric form of CPC remain unclear. To understand this assembly mechanism, we performed an in vitro experiment to study the disassembly and reassembly behaviors of CPC using isolated CPC subunits. The dissociation of the CPC<inf>O77</inf> subunit was performed using a Phenyl-Sepharose column in 20 mM potassium phosphate buffer (pH 6.0) containing 7.0 M urea. For the first time, crystals of isolated CPC subunits were obtained and analyzed after separation. After the removal of urea from the purified α and β subunits, we performed an in vitro reassembly experiment for CPC and analyzed the reconstructed CPC using spectrophotometric and X-ray crystal structure analyses. The crystal structure of the reassembled CPC was nearly identical to that of the original CPC<inf>O77</inf>. The findings of this study indicate that the octameric CPC<inf>O77</inf> is a naturally occurring form in the thermophilic cyanobacterium Thermoleptolyngbya sp. O-77.

DOI： 10.1016/j.jbiosc.2023.12.015

Web of Science

Scopus

PubMed

researchmap

Language：English   Publisher：Chemical Science  

Nicotinamide adenine dinucleotide (NAD⁺) and its reduced form (NADH) are key cofactors serving as essential hydrogen acceptors and donors to facilitate energy and material conversions under mild conditions. We demonstrate direct electrochemical conversion to achieve highly efficient regeneration of enzymatically active 1,4-NADH using a Pt-modified TiO<inf>2</inf> catalyst grown directly on a Ti mesh electrode (Pt-TOT). Spectral analyses revealed that defects formed by the inclusion of Pt species in the lattice of TiO<inf>2</inf> play a critical role in the regeneration process. In particular, Pt-TOT containing approximately 3 atom% of Pt exhibited unprecedented efficiency in the electrochemical reduction of NAD⁺ at the lowest overpotential to date. This exceptional performance led to the production of active 1,4-NADH with a significantly high yield of 86 ± 3% at −0.6 V vs. Ag/AgCl (−0.06 V vs. RHE) and an even higher yield of 99.5 ± 0.4% at a slightly elevated negative potential of −0.8 V vs. Ag/AgCl (−0.2 V vs. RHE). Furthermore, the electrochemically generated NADH was directly applied in the enzymatic conversion of pyruvic acid to lactic acid using lactate dehydrogenase.

DOI： 10.1039/d3sc04104b

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

An H₂-derived energy carrier that stores electrons from H₂ at room temperature and transfers them to CO₂ at leisure at room temperature.

DOI： 10.1039/d3cc05285k

Web of Science

Scopus

PubMed

researchmap

Language：English   Publisher：Bioresource Technology  

Bacterial membranes shield the intracellular compartment by selectively allowing unwanted substances to enter in, which in turn reduces overall catalytic efficiency. This report presents a model system using the isolated plasma membranes of Citrobacter sp. S-77 that harbor oxygen-stable [NiFe]hydrogenase and [Mo]formate dehydrogenase, which are integrated into a natural catalytic nanodevice through an electron transfer relay. This naturally occurring nanodevice exhibited selectivity and efficiency in catalyzing the H<inf>2</inf>-driven conversion of CO<inf>2</inf> to formate with the rate of 817 mmol·L^–1·g<inf>protein</inf>^–1·h⁻¹ under mild conditions of 30 °C, pH 7.0, and 0.1 MPa. When the isolated plasma membranes of Citrobacter sp. S-77 was immobilized with multi-walled carbon nanotubes and encapsulated in hydrogel beads of gellan-gum cross-linked with calcium ions, the catalyst for formate production remained stable over 10 repeated uses. This paper reports the first case of efficient and selective formate production from H<inf>2</inf> and CO<inf>2</inf> using bacterial plasma membranes.

DOI： 10.1016/j.biortech.2023.129921

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

Biocatalytic CO<inf>2</inf> reduction into formate is a crucial strategy for developing clean energy because formate is considered as one of the promising hydrogen storage materials for achieving net-zero carbon emissions. Here, we developed an efficient biocatalytic system to produce formate selectively by coupling two enzymatic activities of H<inf>2</inf> oxidation and CO<inf>2</inf> reduction using encapsulated bacterial cells of Citrobacter sp. S-77. The encapsulated whole-cell catalyst was made by living cells depositing into polyvinyl alcohol and gellan gum cross-linked by calcium ions to form hydrogel beads. Formate production using encapsulated cells was carried out under the resting state conditions in the gas mixture of H<inf>2</inf>/CO<inf>2</inf> (70:30, v/v%). The whole-cell biocatalyst showed highly efficient and selective catalytic production of formate, reaching the specific rate of formate production of 110 mmol L⁻¹· g<inf>protein</inf>⁻¹·h⁻¹ at 30 °C, pH 7.0, and 0.1 MPa. The encapsulated cells can be reused at least 8 times while keeping their high catalytic activities for formate production under mild reaction conditions.

DOI： 10.1016/j.jbiosc.2023.06.002

Web of Science

Scopus

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.jbiosc.2023.06.002. Epub 2023 Jul 1.

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

researchmap

Publisher：Journal of Organometallic Chemistry  

We developed a new method to isolate a natural pigment of mesobiliverdin IXα (MBV) directly from phycocyanin of Thermoleptolyngbya sp. O-77 and synthesized a water-soluble Cp*Ir complex with the isolated MBV ligand. To obtain a high yield of MBV, we modified the conventional method by adding additional two steps: the protease digestion of phycocyanin by trypsin and the isolation of MBV by hydrophobic and silica gel column chromatographies. The Cp*Ir complex with MBV ligand was characterized by electrospray ionization-mass spectrometry, ultraviolet-visible-near-infrared absorption spectroscopy, X-ray photoelectron spectroscopy, ¹H NMR spectroscopy, and H–H correlation spectroscopy. This is the first example of a platinum-group metal complex with MBV as a ligand.

DOI： 10.1016/j.jorganchem.2022.122302

Web of Science

Scopus

Language：English  

Language：English  

researchmap

Publisher：Bulletin of the Chemical Society of Japan  

Current greenhouse gas emissions suggest that keeping global temperature increase below 1.5 degrees, as espoused in the Paris Agreements will be challenging, and to do so, the achievement of carbon neutrality is of utmost importance. It is also clear that no single solution can meet the carbon neutral challenge, so it is essential for scientific research to cover a broad range of technologies and initiatives which will enable the realization of a carbon free energy system. This study details the broad, yet targeted research themes being pioneered within the International Institute for Carbon-Neutral Energy Research (I2CNER). These approaches include hydrogen materials, bio-mimetic catalysts, electrochemistry, thermal energy and absorption, carbon capture, storage and management and refrigerants. Here we outline the state of the art for this suite of technologies and detail how their deployment, alongside prudent energy policy implementation can engender a carbon neutral Japan by 2050. Recognizing that just as no single technological solution will engender carbon neutrality, no single nation can expect to achieve this goal alone. This study represents a recognition of conducive international policy agendas and is representative of interdisciplinary, international collaboration.

DOI： 10.1246/bcsj.20210323

Web of Science

Scopus

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

This paper reports the first example of a reductive C(sp^3)–C(sp^3) homo-coupling of benzyl/allyl halides in aqueous solution by using H_2 as an electron source {turnover numbers (TONs) = 0.5–2.3 for 12 h}. This homo-coupling reaction, promoted by visible light, is catalysed by a water-soluble electron storage catalyst (ESC). The reaction mechanism, and four requirements to make it possible, are also described.

DOI： 10.1039/d1ra08596d

Web of Science

PubMed

CiNii Research

researchmap

Language：Others   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/chem.202102735

Web of Science

PubMed

researchmap

Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/chem.202102735

Language：English  

DOI： 10.1038/s42003-021-02767-x

Web of Science

PubMed

Language：English  

Language：English  

researchmap

Language：English  

DOI： 10.1039/d1cc01611c

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Language：English  

researchmap

Language：English  

DOI： 10.1039/d0cc04789a

Web of Science

PubMed

Language：English   Publisher：American Association for the Advancement of Science (AAAS)  

The study of hydrogenase enzymes (H_2ases) is necessary because of their importance to a future hydrogen energy economy. These enzymes come in three distinct classes: [NiFe] H_2ases, which have a propensity toward H_2 oxidation; [FeFe] H_2ases, which have a propensity toward H_2 evolution; and [Fe] H_2ases, which catalyze H− transfer. Modeling these enzymes has so far treated them as different species, which is understandable given the different cores and ligand sets of the natural molecules. Here, we demonstrate, using x-ray analysis and nuclear magnetic resonance, infrared, Mössbauer spectroscopies, and electrochemical measurement, that the catalytic properties of all three enzymes can be mimicked with only three isomers of the same NiFe complex.

CiNii Research

Language：English  

DOI： 10.1126/sciadv.aaz8181

Web of Science

PubMed

Language：English  

The DNA-binding protein from starved cells (Dps) is found in a wide range of microorganisms, and it has been well characterized. However, little is known about Dps proteins from non-heterocystous filamentous cyanobacteria. In this study, a Dps protein from the thermophilic non-heterocystous filamentous cyanobacterium Thermoleptolyngbya sp. O-77 (TlDps1) was purified and characterized. PAGE and CD analyses of TlDps1 illustrated that it had higher thermostability than previously reported Dps proteins. X-ray crystallographic analysis revealed that TlDps1 possessed His-type ferroxidase centers within the cavity and unique metal binding sites located on the surface of the protein, which presumably contributed to its exceedingly high thermostability.

DOI： 10.1002/2211-5463.12837

Web of Science

PubMed

researchmap

Language：English  

Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium Citrobacter sp. S-77 (CbGAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of CbGAPDH occurred in vivo under sodium hypochlorite (NaOCl) stress. The purified CbGAPDH was highly sensitive to overoxidation by H ₂ O ₂ and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H ₂ O ₂ /NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme in vitro was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected CbGAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of CbGAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.

DOI： 10.1002/2211-5463.12542

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ange.201810270

researchmap

Language：English  

The development of hydrogen fuel cells is greatly hindered by the unwanted generation of H₂O₂ at the cathode. A non-Pt cathode catalyst is now shown to be capable of simultaneously reducing both O₂ and H₂O₂, thus rendering H₂O₂ a useful part of the feed stream. The applicability of this unique catalyst is demonstrated by employing it in a fuel cell running on H₂/CO and O₂/H₂O₂.

DOI： 10.1002/anie.201810270

Web of Science

PubMed

researchmap

Language：English  

Oxidative damage of DNA by reactive oxygen species (ROS) is responsible for aging and cancer. Although many studies of DNA damage by ROS have been conducted, there have been no reports of the oxidation of RNA components, such as guanosine monophosphate, by metal-based species in water. Here, we report the first case of oxidation of guanosine monophosphate to 8-oxoguanosine monophosphate by a metal-based oxygen bound species, derived from O
₂
and in water.

DOI： 10.1002/asia.201801267

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Citrobacter sp. S-77 [NiFe]-hydrogenase harbors a standard [4Fe-4S] cluster proximal to the Ni-Fe active site. The presence of relocatable water molecules and a flexible aspartate enables the [4Fe-4S] to display redox-dependent conformational changes. These structural features are proposed to be the key aspects that protect the active site from O2 attack.

DOI： 10.1039/c8cc06261g

Web of Science

PubMed

researchmap

Language：English  

We report the reactivity of a new Mn^I(cyclam) complex (cyclam: 1,4,8,11-tetraazacyclotetradecane) toward O₂ and H₂O as a model for the photoinhibited species of oxygen-evolving complex (OEC). The reactivity varies according to the number of CO ligands. A Mn^I dicarbonyl complex, [Mn^I(cyclam)(CO)₂]⁺ reacts with O₂, but not with H₂O, to form a bis(μ-oxo)Mn₂ ^III,IV complex, though a Mn^I tricarbonyl complex, [Mn^I(cyclam)(CO)₃]⁺ does not react with either O₂ or H₂O. Newly synthesized Mn^I(cyclam) dicarbonyl complex was characterized by ESI mass spectrometry, UVvis absorption spectroscopy, IR spectroscopy, and X-ray analysis.

DOI： 10.1246/cl.170869

Web of Science

researchmap

Language：English  

We report the mechanistic investigation of catalytic H₂ evolution from formic acid in water using a formate-bridged dinuclear Ru complex as a formate hydrogen lyase model. The mechanistic study is based on isotope-labeling experiments involving hydrogen isotope exchange reaction.

DOI： 10.1080/14686996.2017.1379857

Web of Science

PubMed

researchmap

Language：English  

We report an Ir complex as an anode catalyst capable of switching between a hydrogenase-type fuel-cell mode and a photosystem II-type solar-cell mode. This catalyst is connected to carbon-black-supported platinum as a cathode catalyst, which reduces dioxygen in a manner analogous to cytochrome c oxidase. Together, they make a system capable of switching between the two modes.

DOI： 10.1002/cctc.201700995

Web of Science

researchmap

Language：English  

We present a mechanistic investigation for the activation of H₂ and O₂, induced by a simple ligand effect within [NiFe] models for O₂-tolerant [NiFe]hydrogenase. Kinetic study reveals Michaelis-Menten type saturation behaviors for both H₂ and O₂ activation, which is the same behavior as that found in O₂-tolerant [NiFe]hydrogenase. Such saturation behavior is caused by H₂ complexation followed by heterolytic cleavage of H₂ by an outer-sphere base, resulting in the formation of a hydride species showing hydridic character.

DOI： 10.1021/acs.organomet.7b00471

Web of Science

researchmap

Language：English  

Pyruvate ferredoxin oxidoreductase from Citrobacter sp. S-77 (PFOR_S77) was purified in order to develop a method for acetyl-CoA production. Although the purified PFOR_S77showed high O₂-sensitivity, the activity could be remarkably stabilized in anaerobic conditions. PFOR_S77was effectively immobilized on ceramic hydroxyapatite (PFOR_S77-HA) with an efficiency of more than 96%, however, after encapsulation of PFOR_S77-HA in alginate, the rate of catalytic acetyl-CoA production was highly reduced to 36% when compared to that of the free enzyme. However, the operational stability of the PFOR_S77-HA in alginate hydrogels was remarkable, retaining over 68% initial activity even after ten repeated cycles. The results suggested that the PFOR_S77-HA hydrogels have a high potential for biotechnological application.

DOI： 10.1016/j.biortech.2016.12.051

Web of Science

PubMed

researchmap

Language：English   Publisher：The Chemical Society of Japan  

Structure and reactivity of a Ru-based peroxide complex as a reactive intermediate of O₂-promoted activation of a C-H bond in a Cp∗ ligand
We report the first example of the characterization of a Ru-based peroxide intermediate of O₂-promoted activation of a C-H bond of the pentamethylcyclopentadienyl (Cp∗) ligand. The peroxide complex activates the C-H bond to form a tetramethylfulvene complex. We propose a proton-coupled electron-transfer (PCET) mechanism of the C-H bond activation based on the structures and properties of the peroxide and tetramethylfulvene complexes.

DOI： 10.1246/cl.160909

Web of Science

researchmap

Language：English  

The ability to catalyze the oxidation of both H₂ and CO in one reaction pot would be a major boon to hydrogen technology since CO is a consistent contaminant of H₂ supplies. Here, we report just such a catalyst, with the ability to catalyze the oxidation of either or both H₂ and CO, based on the pH value. This catalyst is based on a NiIr core that mimics the chemical function of [NiFe]hydrogenase in acidic media (pH 4–7) and carbon monoxide dehydrogenase in basic media (pH 7–10). We have applied this catalyst in a demonstration fuel cell using H₂, CO, and H₂/CO (1/1) feeds as fuels for oxidation at the anode. The power density of the fuel cell depends on the pH value in the media of the fuel cell and shows a similar pH dependence in a flask. We have isolated and characterized all intermediates in our proposed catalytic cycles.

DOI： 10.1002/anie.201704864

Web of Science

PubMed

researchmap

Language：English  

Cyanobacteria are widely distributed in marine, aquatic, and terrestrial ecosystems, and play an important role in the global nitrogen cycle. In the present study, we examined the genome sequence of the thermophilic non-heterocystous N₂-fixing cyanobacterium, Thermoleptolyngbya sp. O-77 (formerly known as Leptolyngbya sp. O-77) and characterized its nitrogenase activity. The genome of this cyanobacterial strain O-77 consists of a single chromosome containing a nitrogen fixation gene cluster. A phylogenetic analysis indicated that the NifH amino acid sequence from strain O-77 was clustered with those from a group of mesophilic species: the highest identity was found in Leptolyngbya sp. KIOST-1 (97.9% sequence identity). The nitrogenase activity of O-77 cells was dependent on illumination, whereas a high intensity of light of 40 µmol m^-2 s^-1 suppressed the effects of illumination.

DOI： 10.1264/jsme2.ME17015

Web of Science

PubMed

researchmap

Language：English   Publisher：The Society for Biotechnology, Japan  

Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77
Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE_S77). Interestingly, the ADHE_S77 was unable to be solubilized from membrane with detergents either 1&#37; Triton X-100 or 1&#37; Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH₄)₂SO₄ without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHE_S77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHE_S77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

DOI： 10.1016/j.jbiosc.2015.06.019

Web of Science

PubMed

CiNii Books

researchmap

Other Link： http://search.jamas.or.jp/link/ui/2016330893

Language：English   Publishing type：Research paper (scientific journal)  

The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70Å, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63Å, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60Å resolution, which is considerably better than the 2.00Å resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.

DOI： 10.1107/S2053230X15024152

Web of Science

PubMed

researchmap

Language：English  

NAD⁺-reducing [NiFe] hydrogenases catalyze the oxidoreduction of dihydrogen concomitant with the interconversion of NAD⁺ and NADH. Here, the isolation, purification and crystallization of the NAD⁺-reducing [NiFe] hydrogenase from Hydrogenophilus thermoluteolus TH-1 are reported. Crystals of the NAD⁺-reducing [NiFe] hydrogenase were obtained within one week from a solution containing polyethylene glycol using the sitting-drop vapour-diffusion method and micro-seeding. The crystal diffracted to 2.58Å resolution and belonged to space group C2, with unit-cell parameters a = 131.43, b = 189.71, c = 124.59Å, β = 109.42°. Assuming the presence of two NAD⁺-reducing [NiFe] hydrogenase molecules in the asymmetric unit, V M was calculated to be 2.2ų Da^-1, which corresponds to a solvent content of 43%. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

DOI： 10.1107/S2053230X14026521

Web of Science

PubMed

researchmap

Language：English  

The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ∼ 2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ∼ 0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ∼ 0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O₂ tolerance.

DOI： 10.1016/j.bbabio.2014.06.011

researchmap

Language：English  

We report the decomposition of formic acid to hydrogen and carbon dioxide, catalysed by a NiRu complex originally developed as a [NiFe]hydrogenase model. This is the first example of H₂ evolution, catalysed by a [NiFe]hydrogenase model, which does not require additional energy.

DOI： 10.1039/c4cc05911e

Web of Science

PubMed

researchmap

Language：English  

Herein, we report the first crystal structure of a monomeric p-semiquinonato d-block complex and its reactivity toward dioxygen, closely associated with a biological system of an oxygen evolving centre of photosystem II. This journal is

DOI： 10.1039/c4cc06055e

Web of Science

PubMed

researchmap

Language：English  

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDH_S77) was a monomer with molecular mass of approximately 150kDa. On SDS-PAGE, the purified FDH_S77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87kDa protein band were identical to that of the large subunit of 95kDa, indicating that the purified FDH_S77 consisted of two subunits; a 95kDa large subunit and a 32kDa small subunit. The purified FDH_S77 in this purification did not contain a heme b subunit, but the FDH_S77 showed significant activity for formate oxidation, determined by the V_max of 30.4U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDH_S77 is a molybdenum-containing enzyme, displaying a remarkable O₂-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

DOI： 10.1016/j.jbiosc.2014.03.011

Web of Science

PubMed

researchmap

Language：German   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ange.201404701

researchmap

Language：English  

Reported herein is an electrode for dihydrogen (H₂) oxidation, and it is based on [NiFe]Hydrogenase from Citrobacter sp. S-77 ([NiFe] _S77). It has a 637 times higher mass activity than Pt (calculated based on 1 mg of [NiFe]_S77 or Pt) at 50 mV in a hydrogen half-cell. The [NiFe]_S77 electrode is also stable in air and, unlike Pt, can be recovered 100% after poisoning by carbon monoxide. Following characterization of the [NiFe]_S77 electrode, a fuel cell comprising a [NiFe] _S77 anode and Pt cathode was constructed and shown to have a a higher power density than that achievable by Pt.

DOI： 10.1002/anie.201404701

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

Many types of superoxide dismutases have been purified and characterized from various bacteria, however, a psychrophilic Mn-superoxide dismutase (MnSOD) has not yet been reported. Here, we describe the purification and the biochemical characterization of the psychrophilic MnSOD from Exiguobacterium sp. strain OS-77 (EgMnSOD). According to 16S rRNA sequence analysis, a newly isolated bacterium strain OS-77 belongs to the genus Exiguobacterium. The optimum growth temperature of the strain OS-77 is 20 °C. The EgMnSOD is a homodimer of 23.5 kDa polypeptides determined by SDS-PAGE and gel filtration analysis. UV-Vis spectrum and ICP-MS analysis clearly indicated that the homogeneously purified enzyme contains only a Mn ion as a metal cofactor. The optimal reaction pH and temperature of the enzyme were pH 9.0 and 5 °C, respectively. Notably, the purified EgMnSOD was thermostable up to 45 °C and retained 50 % activity after 21.2 min at 60 °C. The differential scanning calorimetry also indicated that the EgMnSOD is thermostable, exhibiting two protein denaturation peaks at 65 and 84 °C. The statistical analysis of amino acid sequence and composition of the EgMnSOD suggests that the enzyme retains psychrophilic characteristics.

DOI： 10.1007/s00792-013-0621-x

Web of Science

PubMed

researchmap

Other Link： http://link.springer.com/article/10.1007/s00792-013-0621-x/fulltext.html

Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Chemical Society of Japan  

We report the synthesis of mononuclear nonheme manganese(V)oxo complexes in aqueous acetonitrile solution from the reaction of manganese(III) complexes using hydrogen peroxide as an oxidant for the first time. A crystal structure of chloro derivative of manganese(V)oxo complex and its reactivity toward 3,5-di-tert-butyl-catechol are also reported.

DOI： 10.1246/cl.140376

Web of Science

researchmap

Language：English  

A new cyanobacterium of strain O-77 was isolated from a hot spring at Aso-Kuju National Park, Kumamoto, Japan. According to the phylogenetic analysis determined by 16S rRNA gene sequence, the strain O-77 belongs to the genus Leptolyngbya, classifying into filamentous non-heterocystous cyanobacteria. The strain O-77 showed the thermophilic behavior with optimal growth temperature of 55°C. Moreover, we have purified and characterized the oxygen-evolving photosystem II (PSII) from the strain O-77. The O₂-evolving activity of the purified PSII from strain O-77 (PSII_O77) was 1275±255μmol O₂ (mg Chl a)^-1h^-1. Based on the results of MALDI-TOF mass spectrometry and urea-SDS-PAGE analysis, the purified PSII_O77 was composite of the typical PSII components of CP47, CP43, PsbO, D2, D1, PsbV, PsbQ, PsbU, and several low molecular mass subunits. Visible absorption and 77K fluorescence spectra of the purified PSII_O77 were almost identical to those of other purified PSIIs from cyanobacteria. This report provides the successful example for the purification and characterization of an active PSII from thermophilic, filamentous non-heterocystous cyanobacteria.

DOI： 10.1016/j.jbiosc.2014.01.009

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

[NiFeSe]hydrogenases are promising biocatalysts in H₂-based technology due to their high catalytic activity and O₂-stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]_DvMF). The [NiFeSe]_DvMF was composed of two subunits, corresponding to a large subunit of 58.3 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe]hydrogenases having catalytic bias toward H₂-production, the [NiFeSe]_DvMF showed 11-fold higher specific activity of H₂-oxidation (2444 U/mg) than that of H₂-production (217 U/mg). At the optimal reaction temperature of the enzyme (65°C), the specific activity of H₂-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]_DvMF clearly indicated that the enzyme had a remarkable O₂-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]_DvMF. These results indicate that the [NiFeSe]_DvMF can play as a new H₂-oxidizing and O₂-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe]hydrogenases.

DOI： 10.1016/j.jbiosc.2012.10.011

Web of Science

PubMed

researchmap

Language：English  

Hydrogenases are of great interest due to their potential use in H ₂-based technology. However, most hydrogenases are highly sensitive to O ₂, which have been the major bottleneck in hydrogenase studies. Here we report an O ₂-stable membrane-bound [NiFe]hydrogenase (MBH) purified from a newly isolated strain, S-77. According to the 16S rRNA gene sequence and phylogenetic analysis of the strain S-77, it belongs to the genus of Citrobacter. In vitro experiments using the cytoplasmic membrane of strain S-77 suggested that a cytochrome b acts as the physiological electron acceptor of the MBH. The purified MBH was composed of a dimer of heterodimers, consisting of two distinct subunits with the molecular weights of 58.5 and 38.5 kDa. The enzyme showed a specific activity for H ₂-oxidation of 661U/mg, which is 35-fold greater than that for H ₂-production of 18.7U/mg. Notably, the MBH showed a remarkable O ₂-stability, maintaining almost 95% of its original activity even after incubation for 30 h in air at 4°C. These results suggest that the O ₂-stable MBH may play an important role in the H ₂-metabolic pathway under the aerobic conditions of Citrobacter sp. S-77. This is the first report of the purification and biochemical characterization of an O ₂-stable MBH from the genus of Citrobacter.

DOI： 10.1016/j.jbiosc.2012.05.018

Web of Science

PubMed

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Chemical Society (ACS)  

We propose a modified mechanism for the inhibition of [NiFe]hydrogenase ([NiFe]H ₂ase) by CO. We present a model study, using a NiRu H ₂ase mimic, that demonstrates that (i) CO completely inhibits the catalytic cycle of the model compound, (ii) CO prefers to coordinate to the Ru ^II center rather than taking an axial position on the Ni ^II center, and (iii) CO is unable to displace a hydrido ligand from the NiRu center. We combine these studies with a reevaluation of previous studies to propose that, under normal circumstances, CO inhibits [NiFe]H ₂ase by complexing to the Fe ^II center.

DOI： 10.1021/ic200965t

Web of Science

PubMed

researchmap

Language：English  

Membrane-bound respiratory [NiFe] hydrogenase is an H
₂-uptake enzyme found in the periplasmic space of bacteria that plays a crucial role in energy-conservation processes. The heterodimeric unit of the enzyme from Hydrogeno-vibrio marinus was purified to homogeneity using chromatographic procedures. Crystals were grown using the sitting-drop vapour-diffusion method at room temperature. Preliminary crystallographic analysis revealed that the crystals belonged to space group P2
₁, with unit-cell parameters a = 75.72, b = 116.59, c = 113.40 Å, β = 91.3°, indicating that two heterodimers were present in the asymmetric unit.

DOI： 10.1107/S1744309111019804

researchmap

Language：English  

The membrane-bound [NiFe]-hydrogenase from Hydrogenovibrio marinus (HmMBH) was purified homogeneously under anaerobic conditions. Its molecular weight was estimated as 110 kDa, consisting of a heterodimeric structure of 66 kDa and 37 kDa subunits. The purified enzyme exhibited high activity in a wide temperature range: 185 U/mg at 30 °C and 615 U/mg at 85 °C (the optimum temperature). The K_m and k_cat/K_m values for H₂ were, respectively, 12 μM and 8.58 × 10⁷ M^-1 s^-1. The optimum reaction pH was 7.8, but its stability was particularly high at pH 4.0-7.0. Results show that HmMBH was remarkably thermostable and oxygen-resistant: its half-life was 75 h at 80 °C under H₂, and more than 72 h at 4 °C under air. The air-oxidized HmMBH for 72 h showed only weak EPR signals of Ni-B, suggesting a structural feature in which the active center is not easily oxidized.

DOI： 10.1016/j.ijhydene.2011.03.049

researchmap

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

This communication reports the successful merging of the chemical properties of a natural [NiFe]hydrogenase (Desulfovibrio vulgaris Miyazaki F) and our previously reported [NiRu] hydrogenase-mimic. The catalytic activity of both the natural enzyme and the mimic is almost identical, with the exception of working pH ranges, and this allows us to use them simultaneously in the same reaction flask. In such a manner, isotope exchange between D₂ and H₂O could be conducted over an extended pH range (about 2-10) in one pot under mild conditions at ambient temperature and pressure.

DOI： 10.1039/b926061g

Web of Science

PubMed

researchmap

Language：English  

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 H₂ oxidation and evolution were 8.0 and 4.0, respectively, and the activity ratio (H₂ oxidation/H₂ evolution) was 1.61 × 10² at pH 7.0. The optimal temperature was 75 °C. The enzyme was quite stable under air atmosphere (the half-life of activity was c. 48 h at 4 °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 °C. The activity increased up to 2.5-fold during incubation at 50 °C under H₂. 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 × 10⁷ 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 H₂-oxidizing bacteria. This is the first report of catalytic and biochemical characterization of the respiratory MBH from Hydrogenophaga.

DOI： 10.1111/j.1574-6968.2008.01417.x

researchmap

Language：English  

A hydrogen-oxidizing bacterium strain AH-24 was isolated, which was classified in the genus Hydrogenophaga, based on the 16S rRNA gene sequence. The isolate possessed a typical yellow pigment of Hydrogenophaga species. Its closest relative was Hydrogenophaga pseudoflava, but the assimilation profile of sugar compounds resembled that of no species of Hydrogenophaga. The optimum temperature and pH for autotrophic growth were, respectively, 33-35°C and 7.0. Most hydrogenase activity (benzyl viologen reducing activity) was localized in the membrane fraction (MF), but NAD(P)-reducing hydrogenase activity was detected in neither the membrane nor the soluble fractions. Cytochromes b ₅₆₁ and c₅₅₁ were present in MF; both were reduced when hydrogen was supplied to the oxidized MF, suggesting involvement in respiratory H₂ oxidation as electron carriers. Cytochrome b₅₆₁ was inferred to function as the redox partner of the membrane-bound hydrogenase.

DOI： 10.1111/j.1574-6968.2007.00983.x

researchmap

Language：English  

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 H ₂·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.

DOI： 10.1562/2006-01-16-RA-778

researchmap

Language：English  

Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, pyruvate synthase/pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be -514 and -584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately ∼2.0 spins per molecule, compatible with the idea that C. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters. C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum pyruvate synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kinetic measurements indicated that Fd I had ∼2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidum Fds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.

DOI： 10.1074/jbc.M107852200

researchmap

Language：English  

Rubredoxin (Rd) from the moderately thermophilic green sulfur bacterium Chlorobium tepidum was found to function as an electron acceptor for pyruvate ferredoxin oxidoreductase (PFOR). This enzyme, which catalyzes the conversion of pyruvate to acetyl-CoA and CO₂, exhibited an absolute dependence upon the presence of Rd. However, Rd was incapable of participating in the pyruvate synthase or CO₂ fixation reaction of C. tepidum PFOR, for which two different reduced ferredoxins are employed as electron donors. These results suggest a specific functional role for Rd in pyruvate oxidation and provide the initial indication that the two important physiological reactions catalyzed by PFOPJ pyruvate synthase are dependent on different electron carriers in the cell. The UV-visible spectrum of oxidized Rd, with a monomer molecular weight of 6500, gave a molar absorption coefficient at 492 nm of 6.89 mM^-1 cm^-1 with an A₄₉₂/A₂₈₀ ratio of 0.343 and contained one iron atom/molecule. Further spectroscopic studies indicated that the CD spectrum of oxidized C. tepidum Rd exhibited a unique absorption maximum at 385 nm and a shoulder at 420 nm. The EPR spectrum of oxidized Rd also exhibited unusual anisotropic resonances at g = 9.675 and g = 4.322, which is composed of a narrow central feature with broader shoulders to high and low field. The midpoint reduction potential of C. tepidum Rd was determined to be -87 mV, which is the most electronegative value reported for Rd from any source.

DOI： 10.1074/jbc.274.42.29772

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

Language：English  

Enzymatic reactions involving pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, aerobic, chemolithoautotrophic, and hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, were investigated as the CO₂ exchange reaction and CO₂ fixation reaction using ferredoxin isolated from the same organism as a reductant. The reduced ferredoxin was required in the pyruvate synthetic reaction and the 2-oxoglutarate synthetic reaction by a cell extract that had been treated with a PD-10 column to remove the low molecular weight substances. [¹⁴C]Pyruvate and [¹⁴C]2-oxoglutarate were detected as products of pyruvate synthetic and 2-oxoglutarate synthetic reactions, respectively. Further evidence for the operation of pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase was obtained from experiments on CO₂ exchange reactions using the purified enzymes.

Language：English  

Enzymatic reactions involving pyruvate:ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, aerobic, chemolithoautotrophic, and hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, were investigated as the CO₂ exchange reaction and CO₂ fixation reaction using ferredoxin isolated from the same organism as a reductant. The reduced ferredoxin was required in the pyruvate synthetic reaction and the 2-oxoglutarate synthetic reaction by a cell extract that had been treated with a PD-10 column to remove the low molecular weight substances. [¹⁴C]Pyruvate and [¹⁴C]2-oxoglutarate were detected as products of pyruvate synthetic and 2-oxoglutarate synthetic reactions, respectively. Further evidence for the operation of pyruvate: ferredoxin oxidoreductase and 2-oxoglutarate:ferredoxin oxidoreductase was obtained from experiments on CO₂ exchange reactions using the purified enzymes.

researchmap

Language：English  

Pyruvate:ferredoxin oxidoreductase was purified to electrophoretic homogeneity from an aerobic, thermophilic, obligately chemolithoautotrophic, hydrogenoxidizing bacterium, Hydrogenobacter thermophilus TK-6, by precipitation with ammonium sulfate and fractionation by DEAE-Sepharose CL-GB, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The native enzyme had a molecular mass of 135 kDa and was composed of four different subunits with apparent molecular masses of 46, 31.5, 29, and 24.5 kDa, respectively, indicating that the enzyme has an αβγδ-structure. The activity was detected with pyruvate, coenzyme A, and one of the following electron accepters in substrate amounts: ferredoxin isolated from H. thermophilus, FAD, FMN, triphenyltetrazolium chloride, or methyl viologen. NAD, NADP, ana ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective as the electron acceptor. The temperature optimum for pyruvate oxidation was approximately 80°C. The pH optimum was 7.6-7.8. The apparent K(m) values for pyruvate and coenzyme A at 70°C were 3.45 mM and 54 μM, respectively. The enzyme was extremely thermostable under anoxic conditions; the time for a 50% loss of activity (t(50%)) at 70°C was approximately 8 h.

DOI： 10.1007/s002030050443

researchmap

Language：English  

NADH:ferredoxin reductase (EC 1.18.13) and NAD-reducing hydrogenase (EC 1.12.1.2) activities were detected in the cytoplasm of Hydrogenobacter thermophilus TK-6. NADH:ferredoxin reductase activity was detected using metronidazole, an artificial electron acceptor, which reacts specifica1ly with reduced ferredoxin. Soluble NAD-reducing hydrogenase activity was detected after extended preincubation. The lag disappeared when cell-free extract was incubated anaerobically for more than 30 min. The electron transport system of this chemolithoautotrophic bacterium is discussed.

DOI： 10.1016/0378-1097(96)00132-2

researchmap

Language：English   Publishing type：Doctoral thesis  

博士論文

DOI： 10.11501/3127506

CiNii Research

Language：English  

2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK- 6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography. The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa). The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H. thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen). NAD, NADP, and ferredoxins from Chlorella spp. and Clostridium pasteurianum were ineffective. The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80°C, and the time for a 50% loss of activity at 70°C under anaerobic conditions was 22 h. The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8. The apparent K(m) values for 2-oxoglutarate and coenzyme A at 70°C were 1.42 mM and 80 μM, respectively.

DOI： 10.1128/jb.178.11.3365-3368.1996

researchmap

Language：English   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

Purification and characterization of ferredoxin from hydrogenobacter thermophilus strain TK-6
Ferredoxin was purified from cells of Hydrogenohacter thermophilus strain TK-6. Purification was performed aerobically by the addition of octyl-p-glucoside to the buffers. The purified ferredoxin had a molecular mass of 13,000 and contained a [4Fe-4S] cluster. The protein had a long stretch at the N-terminal region; however, the sequence was not similar to the sequences of ferredoxins with a long stretch from Archaehacteria.

DOI： 10.1271/bbb.60.1513

CiNii Books

researchmap

Language：English   Publishing type：Research paper (scientific journal)  

A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-1. The enzyme consists of two polypeptides of 63 and 31 kDa, respectively. The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type [Ni-Fe] hydrogenases from other origins. The thermostability under a hydrogen gas atmosphere is highly stable at 50°C, which is the optimum temperature for the cell growth.

DOI： 10.1271/bbb.59.917

Language：English  

A membrane-bound hydrogenase was purified aerobically by one step using a hydroxyapatite column after solubilization by acetone treatment from a thermophilic hydrogen-oxidizing bacterium, Pseudomonas hydrogenothermophila strain TH-l. The enzyme consists of two polypeptides of 63 and 31 kDa, respectively. The amino-terminal amino acid sequences of both subunits were homologous to membrane-bound type [Ni–Fe] hydrogenases from other origins. The thermostability under a hydrogen gas atmosphere is highly stable at 50°C, which is the optimum temperature for the cell growth.

DOI： 10.1080/bbb.59.917

researchmap

Language：Others  

Event date： 2022.7

Language：English   Presentation type：Oral presentation (general)  

researchmap

Event date： 2022.7

Language：Japanese  

Country：Japan  

Event date： 2021.5

Language：English   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2021.5

Language：English  

Country：Japan  

researchmap

Event date： 2019.5

Language：English   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

Venue：I2CNER, Kyushu University   Country：Japan  

Event date： 2019.5

Language：English  

Venue：I2CNER, Kyushu University   Country：Japan  

researchmap

Event date： 2018.12

Language：English   Presentation type：Oral presentation (general)  

Venue：Yokohama   Country：Japan  

Event date： 2018.12

Language：English  

Venue：Yokohama   Country：Japan  

researchmap

Event date： 2018.11

Language：English   Presentation type：Oral presentation (general)  

Venue：Buyeo, Korea   Country：Korea, Republic of  

Event date： 2018.11

Language：English  

Venue：Buyeo, Korea   Country：Korea, Republic of  

researchmap

Event date： 2018.9

Language：Japanese  

Venue：Kyoto   Country：Japan  

Event date： 2018.9

Language：Japanese  

Venue：Kyoto   Country：Japan  

researchmap

Event date： 2018.9

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：Osaka   Country：Japan  

Event date： 2018.9

Language：Japanese  

Venue：Osaka   Country：Japan  

researchmap

Event date： 2018.7 - 2018.8

Language：English   Presentation type：Oral presentation (general)  

Venue：Sendai   Country：Japan  

Event date： 2018.7 - 2018.8

Language：English  

Venue：Sendai   Country：Japan  

researchmap

Event date： 2018.3

Language：English  

Country：Cambodia  

Event date： 2018.3

Language：English  

Country：Cambodia  

researchmap

Event date： 2017.12

Language：Japanese  

Country：Japan  

Event date： 2017.12

Language：Japanese  

Country：Japan  

researchmap

Event date： 2017.9

Language：Japanese  

Country：Japan  

Event date： 2017.9

Language：Japanese  

Country：Japan  

researchmap

Event date： 2016.11

Language：Japanese  

Country：Japan  

Event date： 2016.11

Language：Japanese  

Country：Japan  

researchmap

Event date： 2016.7

Language：English  

Country：United States  

Event date： 2016.7

Language：English  

Country：United States  

researchmap

Event date： 2016.2

Language：English  

Country：Japan  

Event date： 2016.2

Language：English  

Country：Japan  

researchmap

Event date： 2015.12

Language：Japanese  

Country：Japan  

Event date： 2015.12

Language：Japanese  

Country：Japan  

researchmap

Event date： 2015.7

Language：English  

Country：United States  

Event date： 2015.7

Language：English  

Country：United States  

researchmap

Event date： 2014.12

Language：English  

Country：Japan  

Event date： 2014.12

Language：English  

Country：Japan  

researchmap

Event date： 2014.8

Language：English  

Country：United States  

Event date： 2014.8

Language：English  

Country：United States  

researchmap