Differential effects on N-2 binding and reduction, HD formation, and azidereduction with alpha-195(His)- and alpha-191(Gln)-substituted MoFe proteins of Azotobacter vinelandii nitrogenase

In contrast to the wild-type MoFe protein, neither the alpha -195(Asn) nor the alpha -191(Lys) MoFe protein catalyzed N-2 reduction to NH3, when compl emented with wild-type Fe protein. However, N-2 was bound by the alpha -195 (Asn) MoFe protein and inhibited the reduction of both protons and C2H2. Th e alpha -191(Lys) MoFe protein did not interact with N-2. With the alpha -1 95(Asn) MoFe protein, the N-2-induced inhibition of substrate reduction was reversed by removing the N-2. Surprisingly, even though added H-2 also rel ieved N-2 inhibition of substrate reduction, the alpha -195(Asn) MoFe prote in did not catalyze HD formation under a N-2/D-2 atmosphere. This observati on is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two rea ctions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two alte red MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor w ith the 195(Asn) MoFe protein. The observed reactivity changes are correlat ed with likely structural changes caused by the amino acid substitutions an d provide important details about the interaction(s) of N-2 H-2, D-2, and a zide with Mo-nitrogenase.