Stereospecificity of acetylene reduction catalyzed by nitrogenase.

Abstract

In addition to catalyzing the reduction of dinitrogen to ammonia, the metalloenzyme nitrogenase catalyzes the reduction of a number of alternative substrates, including acetylene (C(2)H(2)) to ethylene (C(2)H(4)) and, in certain cases, to ethane (C(2)H(6)). The stereochemistry of proton addition for C(2)D(2) reduction to C(2)D(2)H(2) catalyzed by the Mo-dependent nitrogenase has been used to probe substrate binding and proton addition mechanisms. In the present work, the C(2)D(2) 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 Gln, Leu, or Ala, the alpha-subunit 69(Gly) residue substituted by Ser, and the alpha-subunit 195(His) residue substituted by Asn. The stereochemistry of proton addition to C(2)D(2) does not correlate with the measured K(m) values for C(2)H(2) reduction, or with the ability of the enzyme to reduce C(2)H(2) by four electrons to yield C(2)H(6). Instead, the electron flux through nitrogenase was observed to significantly influence the ratio of cis- to trans-1,2-C(2)H(2)D(2) formed. Finally, the product distribution observed for reduction of C(2)H(2) in D(2)O is not consistent with an earlier proposed enzyme-bound intermediate. An alternative model that accounts for the stereochemistry of C(2)H(2) reduction by nitrogenase based on a branched reaction pathway and an enzyme-bound eta(2)-vinyl intermediate is proposed.