Studies of the substrate-reducing capabilities of an altered nitrogenase MoFe protein (alpha-195(Gln) instead of alpha-195(His)) from a mutant of Azotobacter vinelandii show, contrary to an earlier report [Kim, C.-H., Newton, W. E., and Dean, D. R. (1995) Biochemistry 34, 2798-2808], that the alpha-195(Gln) MoFe protein can reduce N-2 to NH3 but at a rate that is <2% of that of the wild type. The extent of effective binding of N-2 by this altered MoFe protein, as monitored by the inhibition of H-2 evolution, is markedly increased as temperature is lowered but virtually eliminated at 45 degrees C. This inhibition of H-2 evolution results in an increase in the ATP:2e(-) ratio, i.e., the number of molecules of MgATP hydrolyzed for each electron pair transferred to substrate, from ca. 5 (the wild-type level) at 45 degrees C to nearly 25 at 13 degrees C. Like wild-type nitrogenase, the N-2 inhibition of H-2 evolution reaches a maximum at an Fe protein:MoFe protein molar ratio of ca. 2.5, suggesting that a highly reduced enzyme may not be necessary for N-2 binding. N-2 binding to the alpha-195(Gln) MoFe protein retains a hallmark of the wild type by producing HD under a mixed N-2/D-2 atmosphere. The rate of HD production and the fraction of total electron flow allocated to HD are similar to those for wild-type nitrogenase under the same conditions. However, the electrons forming HD do not come from those normally producing NH3 (as occurs in the wild type) but are equivalent to those whose evolution as H-2 had been inhibited by N-2. N-2 also inhibits C2H2 reduction catalyzed by the alpha-195(Gln) nitrogenase. This inhibition is relieved by added H-2, resulting in a lowering of the elevated ATP:2e(-) ratio to that found under Ar. With solutions of NaCN, which contain both the substrate, HCN, and the inhibitor CN-, reduction of HCN is not impaired with the alpha-195(Gln) nitrogenase, but the inhibition by CN- of total electron flow to substrate, which is observed with the wild-type MoFe protein, is completely absent. Unlike that of the catalyzed reduction of H+, HCN, or C2H2, the extent of azide reduction to either N-2 or N2H4 is markedly decreased (to 5-7% of that of the wild type) with the alpha-195(Gln) nitrogenase. Azide, like N-2, inhibits H-2 evolution and increases the ATP:2e(-) ratio. Both effects are freely reversible and abolished by CO. Added D-2 does not relieve either effect, implying that N-2 produced from N-3(-) is not the inhibitory species. The correlation between the extremely low rates of reduction for both N-2 and azide by the alpha-195(Gln) nitrogenase and their common ability to inhibit H-2 evolution suggests that alpha-histidine-195 may be an important proton conductor to the FeMo cofactor center and specifically required for reduction of these two substrates.
