Differentiation of Acetylene-Reduction Sites by Stereoselective Proton Addition during Azotobacter vinelandii Nitrogenase-Catalyzed C2D 2 Reduction

Abstract

The interactions of acetylene with its binding site(s) on the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C2D2. Specifically, the effects of changing the C2D2 concentration, electron flux, pH, or the individual presence of N2, ethylene, or CO on the formation of both cis- and trans-1,2-ethylene-d2 from C2D2 were measured. A hypothesis, involving two acetylene-reduction sites, was developed to explain the changes observed in the stereoselective protonation during both substrate-concentration-dependent and electron-flux-dependent C 2D2 reduction. One of these sites is a higher-affinity acetylene-binding site that produces only cis-1,2-ethylene-d2 from C2D2. The other is a lower-affinity acetylene-binding site, which produces both cis- and trans-1,2-ethylene-d2. Added N2 specifically inhibited the production of cis-1,2-ethylene-d 2 from C2D2, which indicates that N 2 binds to (and is reduced at) the higher-affinity acetylene-binding site. High concentrations of added ethylene behaved like very high concentrations of acetylene and inhibited both the electron flux flowing through the enzyme and cis-isomer formation. Added CO, at very low concentrations, did not affect the relative distribution of cis- and trans-isomers, indicating a separate CO-binding site. The results of pH-dependence experiments showed that substrate inhibition at high C 2D2 concentrations is enhanced under acidic conditions but is absent under basic conditions and suggest that a low proton flux has a similar impact to that of a low electron flux; both inhibit cis-1,2-ethylene-d2 formation selectively. Apparently, the factors affecting stereoselective protonation during C2D2 reduction could be the same as those that perturb protonation of the FeMo cofactor when acetylene is reduced. The observed nitrogenase-catalyzed production of ethylene-d1 from C2D2 implicates a reversible protonation step in the mechanistic pathway.