Nitrogenase catalyzes the MgATP-dependent reduction of dinitrogen gas to ammonia. In addition to the physiological substrate, nitrogenase catalyzes reduction of a variety of other multiply bonded substrates, such as acetylene, nitrous oxide, and azide. Although carbon monoxide (CO) is not reduced by nitrogenase, it is a potent inhibitor of all nitrogenase catalyzed substrate reductions except proton reduction. Here, we present kinetic parameters for an altered Azotobacter vinelandii MoFe protein for which the alphaGly(69) residue was substituted by serine (Christiansen, J., Cash, V. L., Seefeldt, 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 alphaSer(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 inhibitor of acetylene, nitrous oxide, and azide reduction. These results are interpreted in terms of a two-site model. Site 1 is a high affinity acetylene-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 acetylene reduction assays. Site 2 is a low affinity acetylene-binding site to which CO, dinitrogen, azide, and nitrous oxide also bind. Site 1 and site 2 are proposed to be located in close proximity within a specific 4Fe-4S face of FeMo cofactor.