Site-directed mutagenesis and gene-replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe
proteins in which the alpha-subunit residue-195 position, normally occupied by a
histidine residue, was individually substituted by a variety of other
amino acids. Structural studies have revealed that this
histidine residue is associated with the
FeMo-cofactor binding domain and probably provides an NH-->S hydrogen bond to a central bridging
sulfide located within
FeMo-cofactor. Substitution by a
glutamine residue results in an altered
MoFe protein that binds but does not reduce N2, the physiological substrate. Although N2 is not a substrate for the altered
MoFe protein, it is a potent inhibitor of both
acetylene and
proton reduction, both of which are otherwise effectively reduced by the altered
MoFe protein. This result provides evidence that N2 inhibits
proton and
acetylene reduction by simple occupancy of a common active site. N2 also uncouples
MgATP from
proton reduction catalyzed by the altered
MoFe protein but does so without lowering the overall rate of
MgATP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe
protein to the
MoFe protein that occurs during
nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. Substitution of the alpha-histidine-195 residue by
glutamine also imparts to the altered
MoFe protein hypersensitivity of both its
acetylene reduction and N2 binding to inhibition by CO, indicating that the
imidazole group of the alpha-histidine-195 residue might protect an Fe contained within the
FeMo-cofactor from attack by CO. Finally, comparisons of the catalytic and spectroscopic properties of altered MoFe
proteins produced by various mutant strains suggest that the alpha-histidine-195 residue has a structural role, which serves to keep
FeMo-cofactor attached to the
MoFe protein and to correctly position
FeMo-cofactor within the
polypeptide matrix, such that N2 binding is accommodated.