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The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(I1) and Ni(I1) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(1I) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(I1) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal > H2-uptake activity > Ni-A signal. Similar results were obtained when the protein was treated with Hg(I1). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D . gigas hydrogenase was found to be highly resistant to digestion by proteases.Copper is an essential element for bacterial growth and metabolism but like other heavy metals, it can be very toxic depending on the concentration. In 1954, Schlegel demonstrated a direct inhibition by copper of the hydrogen metabolism of the Knallgas bacteria Hydrogenomonas [l]. In another early study, Booth and Mercer found that sulphate-reducing bacteria were very sensitive to poisoning by Cu(I1) compared with sulphur-oxidizing and iron-oxidizing bacteria 121. The effect of Cu(I1) on the enzyme activity has often been tested with pure preparations of hydrogenases. In most of the cases studied the presence of this metal was inhibitory [3 -131. An exception was the hydrogenase from the green sulfur bacterium Chlorobium limicola which was only slightly inhibited by 0.5 mM Cu(I1) in the H2-uptake assay [14]. Interestingly enough, the hydrogenase purified from Methanobacterium formicicum was reported to contain two atoms of Cu(II)/ molecule besides other transition metals [15].Cypionka and Dilling have observed that the hydrogenase of extracts of Desuljotomaculum orientis was inhibited by incubation with Cu(II), but inhibition did not occur with whole cells [16]. However, we have found that whole cells of Desuljovihrio gigas are sensitive to Cu(I1). These findings prompted us to study the effect of heavy metals on pure hydrogenase isolated from D. gigas. The enzyme isolated from the periplasmic space contains one atom of nickel/molecule [17, 181; in addition it contains 11 or 12 atoms of iron and sulphur [19], arranged in a cluster with three irons and two more clusters of the [4Fe-4S] type [17]. The enzyme, isolated under oxidizing conditions, exists mainly in an inactive form which we have designated 'unready'; it is only reactivated by strong reducing agents over the course of several hours [20]. Aft...

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