Azotobacter vinelandii Nitrogenases Containing Altered MoFe Proteins with Substitutions in the FeMo-Cofactor Environment: Effects on the Catalyzed Reduction of Acetylene and Ethylene

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C2H2, C2H4, CO, and H2. The altered MoFe proteins used were the α-195Asn or α-195Gln MoFe proteins, which have either asparagine or glutamine substituting for α-histidine-195, and the α-191Lys MoFe protein, which has lysine substituting for α-glutamine-191. On the basis of K m determinations, C2H2 was a particularly poor substrate for the nitrogenase containing the α-191Lys MoFe protein. Using C2D2, a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C2D2H2, and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the α-195Asn or the α-191Lys MoFe proteins, which also exhibited the highest rate of ethane production from C2H2. These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C2H4 was not a substrate of the nitrogenase with the α-191Lys MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the α-195Gln MoFe protein, both of which had a low V max and high K m (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the α-195Asn MoFe protein, which exhibited a K m of 48 kPa and a specific activity for C2H6 formation from C2H4 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the α-195Asn MoFe protein produced cis-C2D2H2 when turned over under trans-C2D2H2. These results suggest that the C2H4-reduction site is affected by substitution at residue α-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C2H4 flux-inhibition site is remote from the C2H4-reduction site. Added CO eliminated C2H4 reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C2H4 is not directly connected to C2H4 reduction. Thus,