Carbon Monoxide Chemistry of a-V70I Mo-Nitrogenase: Evidence from EPR- and IR-Monitored Photolysis - or, what a Difference a Methyl Makes

A critical step in the global nitrogen cycle is the conversion of dinitrogen into biologically accessible ammonia. In Nature this is accomplished by the nitrogenase (N2ase) family of enzymes. Carbon monoxide (CO) has long been known as an inhibitor of dinitrogen reduction by N2ase, but it can also be a substrate of the enzyme, when it is catalytically reduced to hydrocarbons. Understanding the CO interactions with N2ases are thus relevant to both dinitrogen fixation and Fischer-Tropsch-like chemistry. Here, the interaction of CO with the a-V70I variant of Azotobacter vinelandii MoFe N2ase was investigated using EPR- and IR- monitored photolysis of bound CO under cryogenic conditions. This was supplemented by further analysis of stopped-flow FT-IR (SF-FT-IR) data under turnover conditions. The a-V70I variant adds a single methyl group close to the active site, and the results show that this inhibits and slows, but does not substantially chemically change, the binding of CO to the FeMo-cofactor. The EPR spectra of both the hi-CO and lo-CO states closely resemble those from the wild-type enzyme. Similarly, the SF-FT-IR spectrum is strikingly similar, with only small shifts in band energies and comparison with data from wild-type enzyme allows better interpretation of the published SF-FT-IR spectra. The extra carbon does, however, impact and inhibit the photochemical release and migration of CO at cryogenic temperature, resulting in novel CO-bound species. In particular, a photolysis product species, termed Hi-1*, with an IR band at 1944 cm-1 may involve CO photochemically migrating on the FeMo-cofactor.