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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