Azotobacter vinelandii vanadium nitrogenase: formaldehyde is a product of catalyzed HCN reduction, and excess ammonia arises directly from catalyzed azide reduction.
The Mo-nitrogenase-catalyzed reduction of both cyanide and azide results in the production of excess NH3, which is an amount of NH3 over and above that expected to be formed from the well-recognized reactions. Several suggestions about the possible sources of excess NH3 have been made, but previous attempts to characterize these reactions have met with either limited (or no) success or controversy. Because V-nitrogenase has a propensity to release partially reduced intermediates, e.g., N2H4 during N2 reduction, it was selected to probe the reduction of cyanide and azide. Sensitive assay procedures were developed and employed to monitor the production of either HCHO or CH3OH (its further two-electron-reduced product) from HCN. Like Mo-nitrogenase, V-nitrogenase suffered electron-flux inhibition by CN- (but was much less sensitive than Mo-nitrogenase), but unlike the case for Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN-. V-Nitrogenase also released more of the four-electron-reduced intermediate, CH3NH2, than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into excess NH3, and under these conditions, substantial amounts of HCHO, but no CH3OH, were detected for the first time. With azide, in contrast to the case for Mo-nitrogenase, both total electron flux and MgATP hydrolysis with V-nitrogenase were inhibited. V-Nitrogenase, unlike Mo-nitrogenase, showed no preference between the two-electron reduction to N2-plus-NH3 and the six-electron reduction to N2H4-plus-NH3. V-Nitrogenase formed more excess NH3, but reduction of the N2 produced by the two-electron reduction of N3(-) was not its source. Rather, it was formed directly by the eight-electron reduction of N3(-). Unlike Mo-nitrogenase, CO could not completely eliminate either cyanide or azide reduction by V-nitrogenase. CO did, however, eliminate the inhibition of both electron flux and MgATP hydrolysis by CN-, but not that caused by azide. These different responses to CO suggest different sites or modes of interaction for these two substrates with V-nitrogenase.
Department of Biochemistry, The Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Karl
K
Fisher
Michael J
MJ
Dilworth
William E
WE
Newton
D000641:Ammonia / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000201:enzymology*; D002248:Carbon Monoxide / Q000494:pharmacology; D002384:Catalysis; D005557:Formaldehyde / Q000378:metabolism*; D006856:Hydrogen Cyanide / Q000378:metabolism*; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction
D001386:Azides; D005557:Formaldehyde; D006856:Hydrogen Cyanide; D000641:Ammonia; D002248:Carbon Monoxide; D009591:Nitrogenase