Fe:S cluster ligands are the only cysteines required for nitrogenase Fe-protein activities

Abstract

Serine substitutions for the five conserved cysteins (residues 38, 85, 97, 132, and 184) have been made in the Azotobacter vinelandii nitrogenase Fe-protein by site-specific mutagenesis. At least moderate levels of enzyme activity (greater than 10% of wild type enzyme) were found for enzymes with serine substitutions at residues 38, 85, and 184; whereas, no activity was detected for enzymes with serines at residues 97 and 132. This is consistent with cysteines 97 and 132 being the four ligands to the Fe:S cluster (two ligands from each of the two identical subunits). Although previous chemical modification studies had implicated these residues as ligands, the earlier results did not portend the new finding that of all the conserved cysteines only these 2 residues are required for a second function of the Fe-protein. Namely, if either cysteine 97 or 132 is replaced, it appears that a functional Fe:S cluster cannot be incorporated into the apo-Fe-protein. The consequence is that these altered Fe-proteins cannot participate either in substrate reduction or in the biosynthesis of FeMo-cofactor, a metallocofactor of the MoFe-protein. These results implicate the Fe:S center of Fe-protein in the biosynthesis mechanism as either a redox partner or Fe:S donor. Additional results suggest that the posttranslational modification of Fe-protein by nifM product is not the insertion of the Fe:S center.