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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 b561 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. [ABSTRACT FROM AUTHOR]

The amino-acid sequence of cytochrome c552 (PH c552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogeno-bacter thermophilus (HT c552), and from a mesophile, Pseudomonas aeruginosa (PA c551). The PH c552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coil periplasm. The overexpressed PH c552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C2221, with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 Å. The crystals diffract X-rays to around 2.1 Å resolution. [ABSTRACT FROM AUTHOR]

Membrane-bound respiratory [NiFe] hydrogenase is an H2-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 Hydrogenovibrio 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 P21, 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. [ABSTRACT FROM AUTHOR]

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability., Brief Summary: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c., (© 2017 The Authors Protein Science published byWiley Periodicals, Inc. on behalf of The Protein Society.)

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 2.58 Å resolution and belonged to space group C2, with unit-cell parameters a=131.43, b=189.71, c=124.59 Å, β=109.42°. Assuming the presence of two NAD+-reducing [NiFe] hydrogenase molecules in the asymmetric unit, VM was calculated to be 2.2 Å3 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.

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

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 degrees 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 b561 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.

The amino-acid sequence of cytochrome c552 (PH c552) from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus, was more than 50% identical to that of cytochrome c from an extreme thermophile, Hydrogenobacter thermophilus (HT c552), and from a mesophile, Pseudomonas aeruginosa (PA c551). The PH c552 gene was overexpressed as a correctly processed holoprotein in the Escherichia coli periplasm. The overexpressed PH c552 has been crystallized by vapour diffusion from polyethylene glycol 4000 pH 6.5. The crystals belong to space group C222(1), with unit-cell parameters a = 48.98, b = 57.99, c = 56.20 A. The crystals diffract X-rays to around 2.1 A resolution.