Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction.
The interactions of acetylene with its binding site(s) on the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C(2)D(2). Specifically, the effects of changing the C(2)D(2) concentration, electron flux, pH, or the individual presence of N(2), ethylene, or CO on the formation of both cis- and trans-1,2-ethylene-d(2) from C(2)D(2) 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(2)D(2) reduction. One of these sites is a higher-affinity acetylene-binding site that produces only cis-1,2-ethylene-d(2) from C(2)D(2). The other is a lower-affinity acetylene-binding site, which produces both cis- and trans-1,2-ethylene-d(2). Added N(2) specifically inhibited the production of cis-1,2-ethylene-d(2) from C(2)D(2), 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(2)D(2) 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-d(2) formation selectively. Apparently, the factors affecting stereoselective protonation during C(2)D(2) 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-d(1) from C(2)D(2) implicates a reversible protonation step in the mechanistic pathway.
Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Jaehong
J
Han
William E
WE
Newton
D000114:Acetylene / Q000737:chemistry*; D016948:Azotobacter vinelandii / Q000201:enzymology*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry; D002384:Catalysis; D004583:Electrons; D004791:Enzyme Inhibitors / Q000737:chemistry; D005030:Ethylenes / Q000096:biosynthesis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D008983:Molybdoferredoxin / Q000737:chemistry; D009584:Nitrogen / Q000737:chemistry; D009591:Nitrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D010084:Oxidation-Reduction; D011522:Protons; D013237:Stereoisomerism; D013379:Substrate Specificity
D004791:Enzyme Inhibitors; D005030:Ethylenes; D008983:Molybdoferredoxin; D011522:Protons; D002248:Carbon Monoxide; C036216:ethylene; D009591:Nitrogenase; D009584:Nitrogen; D000114:Acetylene