記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語  

記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1002/chem.202102735

記述言語：英語  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語  

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

<p>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.</p>

DOI： 10.1246/cl.160909

記述言語：英語  

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

記述言語：英語  

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&#37; 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

記述言語：英語  

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 (NH_4)_2SO_4 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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語  

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&#37;. Initial phases were determined by the single-wavelength anomalous dispersion method using the anomalous signal from the Fe atoms.

DOI： 10.1107/S2053230X14026521

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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&#37; 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

記述言語：英語  

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 &#37; 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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語  

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

記述言語：英語  

[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

記述言語：英語  

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&#37; 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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語  

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

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語  

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.

記述言語：英語  

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&#37; loss of activity (t(50&#37;)) at 70°C was approximately 8 h.

DOI： 10.1007/s002030050443

記述言語：英語  

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

記述言語：英語  

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&#37; 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

記述言語：英語  

Ferredoxin was purified from cells of Hydrogenobacter thermophilus strain TK-6. Purification was performed aerobically by the addition of octyl-β-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 Archaebacteria.

DOI： 10.1271/bbb.60.1513

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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

記述言語：英語  

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

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