ABSTRACT
A likely entry/exit path for nitrogenase substrates, products, and/or protons involves residues α277(Arg), α192(Ser), and α356(Gly), all of which are highly conserved among MoFe proteins from different organisms. The α192(Ser) and α277(Arg) residues form part of a hydrogen-bonded network that also involves α195(His), which interacts with a FeMo cofactor-based sulfide. The terminal amino groups of α277(Arg) are also hydrogen-bonded directly to α281(Tyr), which resides at the surface of the MoFe protein. Individual amino acid substitutions placed at position α277 or α192 resulted in a variety of effects on the catalytic and/or spectroscopic properties of the resulting variant MoFe protein. Of particular interest was the effect of CO on H2 evolution catalyzed by three MoFe protein variants, α277(Cys), α192(Asp), and α192(Glu). All three variants exhibited CO stimulation of H2 evolution under high-electron flux conditions but not under low-electron flux conditions. This observation is best explained by these variants being redox-compromised but only at the most reduced redox states of the MoFe protein. Normally, these states are accessed and operational only under high-electron flux conditions, and the effect of added CO is to prevent access to these most reduced redox states, resulting in a normal rate of catalysis. Furthermore, via correlation of the effect of pH changes on H2 evolution activity for both the wild type and the α277(Cys) MoFe protein variant under argon, with or without 10% CO present, likely pathways for the delivery of a proton to the FeMo cofactor were identified.
