Enzyme-mediated sulfide production for the reconstitution of [2Fe-2S] clusters into apo-biotin synthase of Escherichia coli - Sulfide transfer from cysteine to biotin

We previously showed that biotin synthase in which the (Fe-S) cluster was l abelled with S-34 by reconstitution donates S-34 to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Flour, A. Mejean & A. Marquet (1998) FEES Le tt. 440, 226-230]. We therefore proposed that the source of sulfur was very likely the (Fe-S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme whi ch means that to restore a catalytic activity, the clusters have to be rege nerated. In this report, we show that the NifS protein from Azotobacter vinelandii a nd C-DES from Synechocystis as well as rhodanese from bovine liver can mobi lize the sulfur, respectively, from cysteine and thiosulfate for the format ion of a [2Fe-2S] cluster in the apoprotein of Escherichia coil biotin synt hase. The reconstituted enzymes were as active as the native enzyme. When [S-35]cysteine was used during the reconstitution experiments in the p resence of NifS, labelled ((FeS)-S-35) biotin synthase was obtained. This e nzyme produced [35S]biotin, confirming the results obtained with the S-34-r econstituted enzyme. NifS was also effective in mobilizing selenium from selenocystine to produc e an (Fe-Se) cluster. However, though NifS could efficiently reconstitute holobiotin synthase fro m the apoform, starting from cysteine, these two effecters had no significa nt effect on the turnover of the enzyme in the in vitro assay.