Roles of MoFe protein α-274-histidine, α-276-tyrosine and α-277-arginine residues in Azotobacter vinelandii nitrogenase catalysis

Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions.

Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277^his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277^his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277^his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277^his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment.

When we studied the purified α-276^his and α-274^gin/α-276^his MoFe proteins and compared the results with the data from the α-277^his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis.