Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at α-195(His) (replaced by α-195(Asn) or α-195(Gln)) or at α-191(Gln) (replaced by α-191(Lys)), 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-195(Asn), α-195(Gln), and α-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the α-195(Asn) 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-195(Gln) 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-191(Lys) MoFe protein, the rates of both CH4 and NH3 production were very low, but the rate of NH3 production was higher, and both rates slowly increased with increasing NaCN concentration. A hypothesis, which is based on the varying apparent affinities of the altered MoFe proteins for HCN and CN-, is advanced to explain the higher rate of NH3 production versus the rate of CH4 production and the effect of increasing NaCN concentration on electron flux to products. A new method for CH3NH2 quantification showed that all four MoFe proteins produced CH3NH2. Added CO significantly inhibited both CH4 and NH3 production from HCN with all MoFe proteins except for the α-191(Lys) MoFe protein, which still manifested its very low rate of NH3 production but without CH4 production. All of the MoFe proteins responded differently to the addition of C2H2 to reactions containing NaCN. With the α-195(Asn) MoFe protein, added C2H2 decreased the rates of both CH4 and NH3 production, but the rate of NH3 production decreased much less. C2H2 also exacerbated the inhibition of electron flux. With the α-195(Gln) MoFe protein, added C2H2 decreased the rates of both CH4 and NH3 production substantially and about equally. C2H2 also eliminated the slight decrease in total electron flux that was caused by NaCN. Added C2H2 hardly affected the α-191(Lys) MoFe protein. The effects of adding C2H2 appear related to the different relative affinities of the various MoFe proteins for HCN versus C2H2. Added C2H4 had no effect on HCN reduction with any of the MoFe proteins. The data are consistent with the 'single HCN/CN- binding site' hypothesis [Lowe, D.J., Fisher, K., Thorneley, R.N.F., Vaughn, S.A., and Burgess, B.K. (1989) Biochemistry 28, 8460-8466], imply important roles for both residues, especially α-191(Gln), in the catalysis of HCN reduction, and suggest that different substrates may use different proton delivery routes
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