Variant MoFe proteins of Azotobacter vinelandii: effects of carbon monoxide on electron paramagnetic resonance spectra generated during enzyme turnover.

The resting state of wild-type nitrogenase MoFe protein exhibits an S=3/2 electron paramagnetic resonance (EPR) signal originating from the FeMo cofactor, the enzyme's active site. When nitrogenase turns over under CO, this signal disappears and one (sometimes two) of three new EPR signals, which also arise from the FeMo cofactor, appears, depending on the CO concentration. The appearance and properties of these CO-inducible EPR signals, which were also generated with variant MoFe proteins (alphaR96Q, alphaR96K, alphaQ191K, alphaR359K, alphaR96K/alphaR359K, alphaR277C, alphaR277H, and DeltanifV) that are impacted around the FeMo cofactor, have been investigated. No new CO-induced EPR signals arise from any variant, suggesting that no new CO-binding sites are produced by the substitutions. All variant proteins, except alphaR277H, produce the lo-CO signal; all, except alphaQ191K, produce the hi(5)-CO signal; but only two (alphaR96Q and DeltanifV) exhibit the hi-CO signal. FeMo cofactor's environment clearly dictates which CO-induced EPR signals are generated; however, none of these EPR signals correlate with CO inhibition of H(2) evolution observed with some of these variants. CO inhibition of H(2) evolution is, therefore, due to CO binding to a different site(s) from those responsible for the CO-induced EPR signals. Some resting-state variants have overlapping S=3/2 EPR signals, whose intensities simultaneously decrease under turnover conditions, indicating that all FeMo cofactor conformations are catalytically active. Moreover, these variants produce a similar number of hi(5)-CO signals after turnover under CO to the number of resting-state S=3/2 signals. The FeMo cofactor associated with the hi(5)-CO signal likely contains two bridging CO molecules.