Differential Effects on N2 Binding and Reduction, HD Formation, and Azide Reduction with α-195His- and α-191Gln-Substituted MoFe Proteins of Azotobacter vinelandii Nitrogenase

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Abstract
In contrast to the wild-type MoFe protein, neither the α-195Asn nor the α-191Lys MoFe protein catalyzed N2 reduction to NH3, when complemented with wild-type Fe protein. However, N2 was bound by the α-195Asn MoFe protein and inhibited the reduction of both protons and C2H2. The α-191Lys MoFe protein did not interact with N2. With the α-195Asn MoFe protein, the N2-induced inhibition of substrate reduction was reversed by removing the N2. Surprisingly, even though added H2 also relieved N2 inhibition of substrate reduction, the α-195Asn MoFe protein did not catalyze HD formation under a N2/D2 atmosphere. This observation is the first indication that these two reactions have different chemical origins, prompting a revision of the current hypothesis that these two reactions are consequences of the same nitrogenase chemistry. A rationale that accounts for the dichotomy of the two reactions is presented. The two altered MoFe proteins also responded quite differently to azide. It was a poor substrate for both but, in addition, azide was an electron-flux inhibitor with the 195Asn MoFe protein. The observed reactivity changes are correlated with likely structural changes caused by the amino acid substitutions and provide important details about the interaction(s) of N2, H2, D2, and azide with Mo-nitrogenase.