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The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named Cu(A) and Cu(Z). Cu(Z) could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)=2.015, A(x)=1.5 mT, g(y)=2.071, A(y)=2 mT, g(z)=2.138, A(z)=7 mT) and a strong absorption at approximately 640 nm. Cu(A) can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x)=g(y)= 2.021, A(x) = A(y)=0 mT, g(z) = 2.178, A(z)= 4 mT) and absorption bands at 480, 540, and approximately 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the Cu(A) center. In form A, Cu(A) is predominantly oxidized (S = (1)/(2), Cu(1.5+)-Cu(1.5+)), while in form B it is mostly in the one-electron reduced state (S = 0, Cu(1+)-Cu(1+)). In both forms, Cu(Z) remains reduced (S = 1/2). Complete crystallographic data at 2.4 A indicate that Cu(A) is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu(Z) is a novel tetracopper cluster [Brown, K., et al. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g x 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(II1). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and x 2.0 (component I) and at 2.33, 2.24, and x 2.0 (component 11). In the reduced state, after exposure to an Hz atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers : center I (2.03, 1.89 and 1.86) detectable below 10 K, and center I1 (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 89421. During the course of a redox titration at pH 7.6 using Hz gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fastrelaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity. This indicates that the redox state of the isolated enzyme is important for the full expression of enzymatic activity. The catalytic properties were also followed by the proton-deuterium exchange reaction. The isolated hydrogenases produced Hz/HD ratios higher than those observed for nonselenium-containing hydrogenases.The enzyme responsible for the biological activation of H2, termed hydrogenase [l, 21, has a central role in many relevant anaerobic processes where molecular hydrogen is oxidized or evolved. Also, molecular hydrogen, via the hydrogenase system, is a link between different bacterial consortia w...

SUMMARYThree types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical ironsulfur center believed to be involved in the activation of H E. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensi-Correspondence to: G. Fauque, Section Enzymologie et Biochimie Bact6rienne, ARBS, CEN Cadarache, 13108 Saint-Paul-Lez-Durance Cedex, France. tive to CO and NO 2. It is not present in all species of Desulfovibrio.The nickel-(iron-sulfur)-containing hydrogenases ([NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO 2 . The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(ironsulfur)-selenium-containing hydrogenases ([NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of 0168-6445/88/$03.50

Three types of hydrogenases have been isolated from the sulfate‐reducing bacteria of the genus Desulfobibrio. They differ in their subunit and metal compositions, physico‐chemical characteristics, amino acid sequences, immunological ractivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as ‘iron only’ hydrogenases and nickel‐containing hydrogenases. The iron‐sulfur‐containg hydrogenase ([Fe] hydrogenase) contains two ferredoxin‐type (4Fe‐4S) clusters and an atypical iron‐sulfur center belived to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton‐deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2−. It is not present in all species of Desulfovibrio The nickel‐(iron‐sulfur)‐containing hydrogenases ([NiFe] hydrogenase) posses two (4Fe‐4S) centers and one (3Fe‐xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2−. The genes encoding the large and small subunits of a periplasmic and a membrane‐bound species of the [NiFe] hydrogenase have been cloned in Eschierichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal‐binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel‐iron‐sulfur)‐selenium‐containing hydrogenases ([NiFe‐Se] hydrohenases) which contain nickel and selenium in equimoleular amounts plus (4Fe‐4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfrovibio (D) baculatus (DSM 1743) (for abbrviations see appendix) have been cloned

In this review, we focus on the activities transpiring in the anaerobic segment of the sulfur cycle occurring in the gut environment where hydrogen sulfide is produced. While sulfate-reducing bacteria are considered as the principal agents for hydrogen sulfide production, the enzymatic desulfhydration of cysteine by heterotrophic bacteria also contributes to production of hydrogen sulfide. For sulfate-reducing bacteria respiration, molecular hydrogen and lactate are suitable as electron donors while sulfate functions as the terminal electron acceptor. Dietary components provide fiber and macromolecules that are degraded by bacterial enzymes to monomers, and these are fermented by intestinal bacteria with the production to molecular hydrogen which promotes the metabolic dominance by sulfate-reducing bacteria. Sulfate is also required by the sulfate-reducing bacteria, and this can be supplied by sulfate- and sulfonate-containing compounds that are hydrolyzed by intestinal bacterial with the release of sulfate. While hydrogen sulfide in the intestinal biosystem may be beneficial to bacteria by increasing resistance to antibiotics, and protecting them from reactive oxygen species, hydrogen sulfide at elevated concentrations may become toxic to the host.

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A novel obligately anaerobic, non-spore-forming, rod-shaped mesophilic bacterium, which stained Gram-positive but showed the typical cell wall structure of Gram-negative bacteria, was isolated from an upflow anaerobic filter treating abattoir wastewaters in Tunisia. The strain, designated LIND7HT, grew at 20–45 °C (optimum 35–40 °C) and at pH 5.0–8.5 (optimum pH 6.5–7.5). It did not require NaCl for growth, but was able to grow in the presence of up to 2 % NaCl. Sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and nitrite were not used as terminal electron acceptors. Strain LIND7HT used cellobiose, glucose, lactose, mannose, maltose, peptone, rhamnose, raffinose, sucrose and xylose as electron donors. The main fermentation products from glucose metabolism were lactate, acetate, butyrate and isobutyrate. The predominant cellular fatty acids were anteiso-C15 : 0, C15 : 0, C17 : 0 2-OH and a summed feature consisting of C18 : 2ω6,9c and/or anteiso-C18 : 0, and the major menaquinones were MK-9, MK-9(H2) and MK-10. The G+C content of the genomic DNA was 41.4 mol%. Although the closest phylogenetic relatives of strain LIND7HT were Parabacteroides merdae , Parabacteroides goldsteinii and Parabacteroides gordonii , analysis of the hsp60 gene sequence showed that strain LIND7HT was not a member of the genus Parabacteroides . On the basis of phylogenetic inference and phenotypic properties, strain LIND7HT ( = CCUG 60892T = DSM 23697T = JCM 16313T) is proposed as the type strain of a novel species in a new genus within the family Porphyromonadaceae , Macellibacteroides fermentans gen. nov., sp. nov.