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...

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

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Dissimilatory nitrite reduction, carried out by hexaheme proteins, gives ammonia as the final product. Representatives of this enzyme group from 3 bacterial species can also reduce NO to either ammonia or NzO. The redox regulation of the nitrite/nitric oxide activities is discussed in the context of the denitrifying pathway, [25]. Two pairs of magneti~~ly interacting hemes (low-spin/low-spin; low-spinlhighspin) operate in this complex, The high-spin heme binds NO and appears to be the enzyme-active site. Reaction of nitrite with fuIly reduced enzyme reoxidizes the lowspin hemes, but the EPR spectrum reveals persistence of the high-spin heme in the NO-bound form. Since the six-electron reduction of nitrite yields no NO as a free intermediate [25,26], NO appears to exist as an enzymebound and not as a gaseous product during the nitriteto-ammonia transit.Ammonia-generating nitrite reductases have typically been purified from spinach and display a complex active site comprised of a siroheme coupled to a single {4Fe,4S] center fl9]. Nitrite reduction by hexaheme enzymes from strictly and facultatively anaerobic bacteria, such as Desuvovibrio desulfuricans (ATTC 27774) [20], Woiinellu succinogenes [21j, Escherichia co& [223, and V. jkcfzeri [24], also give rise directly to ammonia. In concert, EPR andIn view of such involvement of NO with the hexaheme nitrite reductases, we have studied the capacity of representatives of this group to reduce free NO. Their influence in the pathway, NOz-to Nz is discussed as is the redox regulation of the nitrite/nitric oxide reduction activities in these multi-heme systems. MATERIALS AND METHODS

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.The biological role of nickel is now well established in several and diversified biological systems (1) with relevance to hydrogen metabolism. Nickel-containing hydrogenases have been shown to possess a distinctive coordination environment of the metal center. Extended x-ray absorption fine structure (EXAFS) measurements suggested a predominant sulfur coordination around the monomeric nickel (2). However, so few monomeric thiolate compounds are currently available that they cannot be used for modeling the active site of bacterial hydrogenases (3).Rubredoxin is the simplest protein of the iron-sulfur class; it contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. The simple constitution of this active center as well as the low molecular mass makes this protein suitable for the synthesis of metalsubstituted derivatives.We have successfully substituted the native iron atom of rubredoxins from Desulfovibrio with cobalt and nickel (4). The just formed nickel and cobalt cores were then extensively characterized by a variety of spectroscopic probes including UV/visible, electron paramagnetic resonance, 1H nuclear magnetic resonance, and magnetic circular dichroism (4,5). These techniques indicated that a tetrahedral sulfur coordination was maintained in both Co-and Ni-substituted rubredoxins.In this article we show that the nickel-substituted rubredoxins from three species of sulfate-reducing bacteria of the genus Desulfovibrio are active in both the deuterium-proton exchange reaction and in H2 production (using dithionitereduced methyl viologen as electron donor). The nickelsubstituted rubredoxins, providing a sulfur environment for the metal center, can mimic in some aspects the bacterial hydrogenase activity. MATERIAL AND METHODSPreparation of Nickel-Substituted Rubredoxins. The growth conditions on a lactate/sulfate medium for Desulfovibrio gigas (NCIB 9332) and Desulfovibrio desulfuricans strains Berre-eau (NCIB 8387) and ATCC 27774 as well as the purification of their rubredoxins have been reported (6-9).The experimental procedure used for the preparation of the aporubredoxin and the reconstitution of the active center with nickel is described elsewhere (4, 5). The apoprotein was prepared by precipitation with trichloroacetic acid and reconstituted by adding a stoichiometric amount of nickel(II) nitrate. The nickel-substituted protein was then purified by gel filtration. Metal analyses were performed by plasma emission spectroscopy with a Jarrell-Ash model 750 Atomcomp. Protein concentrations were determined by the Lowry method with bovine serum albumin as a standard (10) or by using the previously determined extinction coefficient (448nm = 3200 M-1 cm-1) ...

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The activation of the periplasmic (NiFe) hydrogenase from Desufivibrio gigas by dihydrogen is a complex phenomenon involving both 'slow' and 'fast' reactions. Carbon monoxide, a competitive inhibitor of hydrogenase activity, is demonstrated to cause the slow activation nearly as well as dihydrogen. Carbon monoxide does not reduce the (NiFe) hydrogenase and the fast reductive activation is effected by deuterium in the exchange assay. In the presence of dithionite, which immediately reduces the redox centers of the (NiFe) hydrogenase, the slow activation is still essential to attain full activity. Thus, the slow non-reductive and fast reductive steps of the activation can occur in any sequence.