EVIDENCE FOR MULTIPLE SUBSTRATE-REDUCTION SITES AND DISTINCT INHIBITOR-BINDING SITES FROM AN ALTERED AZOTOBACTER-VINELANDII NITROGENASE MOFE PROTEIN

The arginine-277 residue of the alpha-subunit of the nitrogenase MoFe protein was targeted for substitution because it is (i) a close neighb or of alpha-cysteine-275, which is one of only two residues anchoring the FeMo cofactor to the polypeptide, and (ii) a component of a potent ial channel for entry/exit of substrates/ products and for accepting F eMo cofactor during MoFe-protein maturation. Several of the eight muta nt strains constructed were capable of good diazotrophic growth and al so contained FeMo cofactor as indicated by its biologically unique S = 3/2 EPR spectrum. These observations indicate that the positively cha rged alpha-arginine-277 residue is not required for acceptance of the negatively charged FeMo cofactor by the separately synthesized, cofact or-deficient, apo-MoFe protein. The wide range of nitrogen-fixation ph enotypes shown by these mutant strains generally correlated well with their C2H2- and proton-reduction activities, which range from 5 to 65% of wild-type activity. One notable exception is the histidine-substit uted strain, DJ788 (alpha-277(His)). This strain, although unable to f ix N-2 and grow diazotrophically, elaborates an altered alpha-277(His) MoFe protein that catalyzes the reduction of the alternative substrat es, C2H2, HCN, HN3, and protons. These observations are best explained if multiple redox levels are available to the MoFe protein but the al pha-277(His) MoFe protein is incapable of reaching the more-reduced re dox levels required for nitrogen fixation. Under nonsaturating CO conc entrations, the alpha-277(His) MoFe-protein-catalyzed reduction of C2H 2 showed sigmoidal kinetics, which is consistent with inhibitor-induce d cooperativity among two C2H4-evolving sites and indicates the presen ce of three sites, which can be simultaneously occupied, on the MoFe p rotein, Similar kinetics were not observed for alpha-277(His) MoFe-pro tein-catalyzed reduction of either HCN or HN3 with nonsaturating CO le vels, indicating that these substrates are unlikely to share common bi nding sites with C2H2. Further CN- did not induce cooperativity in C2H 2 reduction and, therefore, CO and CN- are unlikely to share a common binding site. These changed substrate specificities, reinforced by cha nges in the FeMo-cofactor-derived S = 3/2 EPR spectrum, clearly indica te the importance of the alpha-277 residue in catalysis and the delica te control exerted on the properties of bound FeMo cofactor by its pol ypeptide environment.