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Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox-dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non-invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase-mediated reactions simultaneously. Using the proposed system, we confirmed that H2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D2 /H2 O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction (k) at the active site and product release rate (kout ) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.

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Hydrogenases are microbial enzymes which catalyze uptake and production of H(2). Hydrogenases are classified into 10 classes based on the electron carrier specificity, or into 3 families, [NiFe]-family (including [NiFeSe]-subfamily), [FeFe]-family and [Fe]-family, based on the metal composition of the active site. H(2) is heterolytically cleaved on the enzyme (E) to produce EH(a)H(b), where H(a) and H(b) have different rate constants for exchange with the medium hydron. X-ray crystallography unveiled the three-dimensional structures of hydrogenases. The simplest [NiFe]-hydrogenase is a heterodimer, in which the large subunit bears the Ni-Fe center buried deep in the protein, and the small subunit bears iron-sulfur clusters, which mediate electron transfer between the Ni-Fe center and the protein surface. Some hydrogenases have additional subunit(s) for interaction with their electron carriers. Various redox states of the enzyme were characterized by EPR, FTIR, etc. Based on the kinetic, structural and spectroscopic studies, the catalytic mechanism of [NiFe]-hydrogenase was proposed to explain H(2)-uptake, H(2)-production and isotopic exchange reactions.(Communicated by Shigekazu NAGATA, M.J.A.).

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Erabutoxins a and b are neurotoxins isolated from venom of a sea snake Laticauda semifasciata (erabu-umihebi). Amino acid sequences of the toxins indicated that the toxins are members of a superfamily consisting of short and long neurotoxins and cytotoxins found in sea snakes and terrestrial snakes. The short neurotoxins to which erabutoxins belong act by blocking the nicotinic acetylcholine receptor on the post synaptic membrane in a manner similar to that of curare. X-ray crystallography and NMR analyses showed that the toxins have a three-finger structure, in which three fingers made of three loops emerging from a dense core make a gently concave surface of the protein. The sequence comparison and the location of essential residues on the protein suggested the mechanism of binding of the toxin to the acetylcholine receptor. Classification of snakes by means of sequence comparison and that based on different morphological features were inconsistent, which led the authors to propose a hypothesis "Evolution without divergence."

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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 D(2) and H(2)O could be conducted over an extended pH range (about 2-10) in one pot under mild conditions at ambient temperature and pressure.

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The pH-dependent hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes and hydrogenation of the carbonyl compounds have been investigated with water-soluble bis(mu-thiolate)(mu-hydride)NiRu complexes, Ni(II)(mu-SR)(2)(mu-H)Ru(II) {(mu-SR)(2) = N,N'-dimethyl-N,N'-bis(2-mercaptoethyl)-1,3-propanediamine}, as functional models for [NiFe]hydrogenases. In acidic media (at pH 4-6), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes has H(+) properties, and the complexes catalyse the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes. A mechanism of the hydrogen isotope exchange reaction between gaseous isotopes and medium isotopes through a low-valent Ni(I)(mu-SR)(2)Ru(I) complex is proposed. In contrast, in neutral-basic media (at pH 7-10), the mu-H ligand of the Ni(II)(mu-SR)(2)(mu-H)Ru(II) complexes acts as H(-), and the complexes catalyse the hydrogenation of carbonyl compounds.

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Hexadecaheme high molecular weight cytochrome c from a sulfate-reducing bacterium, Desulfovibrio vulgaris Miyazaki F has been successfully purified and crystallized. X-ray diffraction data have been collected by the multiple wavelength anomalous dispersion method. The crystal belongs to the space group P2(1)2(1)2(1) with unit-cell parameters a=60.42, b=84.29 and c=144.16 A and contains one molecule per asymmetric unit.

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The carbon monoxide complex of [NiFe]hydrogenase from Desulfovibrio vulgaris Miyazaki F has been characterized by X-ray crystallography and absorption and resonance Raman spectroscopy. Nine crystal structures of the [NiFe]hydrogenase in the CO-bound and CO-liberated forms were determined at 1.2-1.4 A resolution. The exogenously added CO was assigned to be bound to the Ni atom at the Ni-Fe active site. The CO was not replaced with H(2) in the dark at 100 K, but was liberated by illumination with a strong white light. The Ni-C distances and Ni-C-O angles were about 1.77 A and 160 degrees, respectively, except for one case (1.72 A and 135 degrees ), in which an additional electron density peak between the CO and Sgamma(Cys546) was recognized. Distinct changes were observed in the electron density distribution of the Ni and Sgamma(Cys546) atoms between the CO-bound and CO-liberated structures for all the crystals tested. The novel structural features found near the Ni and Sgamma(Cys546) atoms suggest that these two atoms at the Ni-Fe active site play a role during the initial H(2)-binding process. Anaerobic addition of CO to dithionite-reduced [NiFe]hydrogenase led to a new absorption band at about 470 nm ( approximately 3000 M(-1)cm(-1)). Resonance Raman spectra (excitation at 476.5 nm) of the CO complex revealed CO-isotope-sensitive bands at 375/393 and 430 cm(-1) (368 and 413 cm(-1) for (13)C(18)O). The frequencies and relative intensities of the CO-related Raman bands indicated that the exogenous CO is bound to the Ni atom with a bent Ni-C-O structure in solution, in agreement with the refined structure determined by X-ray crystallography.

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