Azotobacter vinelandii Nitrogenases with Substitutions in the FeMo-Cofactor Environment of the MoFe Protein:  Effects of Acetylene or Ethylene on Interactions with H+, HCN, and CN-

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at α-195His (replaced by α-195Asn or α-195Gln) or at α-191Gln (replaced by α-191Lys), were used to probe the interactions of HCN and CN-, both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C2H2 enhances the rate of CH4 production from HCN reduction by wild-type nitrogenase. In the absence of C2H2, wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH4 and NH3 production increased with increasing NaCN concentration. The NH3 production rate exceeded the CH4 production rate up to 5 mM NaCN, at which point they became equal. The “excess NH3” likely arises from the two-electron reduction of HCN to CH2NH, some of which is released and hydrolyzed to HCHO plus NH3. With added C2H2, the rate of CH4 production increased but only until it equaled that of NH3 production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C2H2. The increased CH4 production did not arise from the added C2H2. The lowered total electron flux with C2H2 present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH2NH would be displaced, more CH2NH would undergo the full six-electron reduction, and the rate of CH4 production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the α-subunit of the MoFe protein at positions α-191 and α-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH4 production by the α-195Asn, α-195Gln, and α-191Lys MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the α-195Asn MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH4 and NH3 production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the α-195Gln MoFe protein, the rates of production of both CH4 and NH3 were equal at all NaCN concentrations, and total electron flux was hardly affected by changing the NaCN concentration. With the α-191Lys MoFe protein, the rates of both CH4 and NH3 production were very low, but the rate of