Competitive substrate and inhibitor interactions at the physiologically relevant active site of nitrogenase

Nitrogenase catalyzes the MgATP-dependent reduction of dinitrogen gas to am monia. In addition to the physiological substrate, nitrogenase catalyzes re duction: of a variety of other multiply bonded substrates, such as acetylen e, nitrous oxide, and azide, Although Carbon monoxide (CO) is not reduced b y nitrogenase, it is a potent inhibitor of all nitrogenase catalyzed substr ate: reductions except proton reduction. Here, we present kinetic parameter s for an altered Azotobacter vinelandii MoFe protein for which the alpha Gl y(69) residue was substituted by serine (Christiansen, J., Cash, V. L., See feldt, L. C., and Dean, D. R. (2000) J. Biol. Chem. 275, 11459-11464), For the wild type enzyme, CO and acetylene:are: both noncompetitive inhibitors of dinitrogen reduction. However, for the (alpha Ser(69) MoFe protein both CO and acetylene have become competitive inhibitors of dinitrogen reduction . CO is also converted from a noncompetitive inhibitor to a competitive inh ibitor of acetylene, nitrous oxide, and azide reduction. These results are interpreted in terms of a two-site model. Site 1 is a high affinity acetyle ne-binding site to which CO also binds) but dinitrogen, azide, and nitrous oxide do not bind. This site is the one primarily accessed during typical:a cetylene reduction assays. Site 2 is a low affinity acetylene-binding site to which CO, dinitrogen, azide, and nitrous oxide also bind. Site 1 and sit e 2 are proposed to be located in close proximity within a specific 4Fe-4S face of FeMo cofactor.