Tn addition to catalyzing the reduction of dinitrogen to ammonia, the metalloenzyme nitrogenase catalyzes the reduction of a number of alternative substrates, including acetylene (C2H2) to ethylene (C2H4) and, in certain cases, to ethane (C2H6) The stereochemistry of proton addition for C2D2 reduction to C2D2H2? catalyzed by the Mo-dependent nitrogenase has been used to probe substrate binding and proton addition mechanisms. In the present work, the C2D2 reduction stereospecificity of altered MoFe proteins having amino acid substitutions within the active site FeMo-cofactor environment was examined by Fourier transform infrared (FTIR) spectroscopy. Altered MoFe proteins examined included those having the alpha -subunit 96(Arg) residue substituted by Gin, Leu, or Ala, the cr-subunit 69(Gly) residue substituted by Ser, and the cr-subunit 195(His) residue substituted by Asn. The stereochemistry of proton addition to C2D2 does not correlate with the measured K-m values for C2H2 reduction, or with the ability of the enzyme to reduce C2H2 by four electrons to yield C2H6. Instead, the electron flux through nitrogenase was observed to significantly influence the ratio of cis- to trans-1,2-C2H2D2 formed. Finally, the product distribution observed for reduction of C2H2 in D2O is not consistent with an earlier proposed enzyme-bound intermediate. An alternative model that accounts for the stereochemistry of C2H2 reduction by nitrogenase based on a branched reaction pathway and an enzyme-bound eta (2)-vinyl intermediate is proposed.