Stereospecificity of acetylene reduction catalyzed by nitrogenase

Tn addition to catalyzing the reduction of dinitrogen to ammonia, the metal loenzyme nitrogenase catalyzes the reduction of a number of alternative sub strates, including acetylene (C2H2) to ethylene (C2H4) and, in certain case s, to ethane (C2H6) The stereochemistry of proton addition for C2D2 reducti on to C2D2H2? catalyzed by the Mo-dependent nitrogenase has been used to pr obe 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 exa mined by Fourier transform infrared (FTIR) spectroscopy. Altered MoFe prote ins examined included those having the alpha -subunit 96(Arg) residue subst ituted by Gin, Leu, or Ala, the cr-subunit 69(Gly) residue substituted by S er, and the cr-subunit 195(His) residue substituted by Asn. The stereochemi stry of proton addition to C2D2 does not correlate with the measured K-m va lues for C2H2 reduction, or with the ability of the enzyme to reduce C2H2 b y four electrons to yield C2H6. Instead, the electron flux through nitrogen ase 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 interme diate. An alternative model that accounts for the stereochemistry of C2H2 r eduction by nitrogenase based on a branched reaction pathway and an enzyme- bound eta (2)-vinyl intermediate is proposed.