ROLE OF THE IRON MOLYBDENUM COFACTOR POLYPEPTIDE ENVIRONMENT IN AZOTOBACTER-VINELANDII MOLYBDENUM NITROGENASE CATALYSIS

Altered molybdenum-dependent nitrogenases have been constructed with single amino-acid substitutions within targeted regions of the MoFe protein alpha-subunit. Some of these altered non-N2-fixing MoFe proteins, particularly with substitutions at alpha-glutaminyl-191 and alpha-histidinyl-195, exhibit concomitantly altered substrate specificity and a changed S=3/2 electron paramagnetic resonance (EPR) signal, confirming an intimate involvement of FeMo-cofactor with a substrate-binding site within the alpha-subunit. Some substitutions result in catalyzed acetylene reduction that yields both ethylene and ethane, which, although reminiscent of the vanadium-dependent nitrogenase, occurs by a different mechanism. Certain mutant strains having substitutions at either alpha-Gln-191 or alpha-His-195 exhibit H+-reduction ability that, unlike wild type, is sensitive to either CO or N2 or both. These results, together with the recent MoFe-protein structural data, suggest that these residues might fine-tune the electronic properties of FeMo-cofactor and so impact its substrate-reduction capabilities. Also, consistent with the recent structural data, all substitutions at those residues that are now known to bind FeMo-cofactor directly, i.e., the Fe-ligating Cys-275 and the Mo-ligating His-442, produce mutant strains, which are Nif and EPR-silent.