Azotobacter vinelandiiVanadium Nitrogenase:  Formaldehyde Is a Product of Catalyzed HCN Reduction, and Excess Ammonia Arises Directly from Catalyzed Azide Reduction

Abstract

The nitrogenase-catalyzed reduction of both cyanide and azide results in the production of "excess NH 3 ", which is an amount of NH 3 over and above that expected to be formed from the wellrecognized reactions. Several suggestions have been made as to the possible sources of "excess NH 3 ", 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., N 2 H 4 during N 2 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 CH 3 OH (its further two-electron-reduced product) from HCN. Like Monitrogenase, V-nitrogenase suffered electron-flux inhibition by CN − (but was much less sensitive than Mo-nitrogenase) but, unlike Mo-nitrogenase, MgATP hydrolysis was also inhibited by CN − . V-nitrogenase also released more of the four-electron-reduced intermediate, CH 3 NH 2 , than did Mo-nitrogenase. At high NaCN concentrations, V-nitrogenase directed a significant percentage of electron flux into "excess NH 3 " and, under these conditions, substantial amounts of HCHO, but no CH 3 OH, were detected for the first time. With azide, in contrast to 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 N 2 -plus-NH 3 and the six-electron reduction to N 2 H 4 -plus-NH 3 . V-nitrogenase formed more "excess NH 3 ", but reduction of the N 2 produced by the two-electron reduction of N 3 − was not its source. Rather, it was formed directly by the eight-electron reduction of N 3 − . 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. † Support from the National Institutes of Health (Grant #DK 37255 to W.E.N.) is gratefully acknowledged. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2015 January 16. Azotobacter vinelandii is a strictly aerobic N 2 -fixing organism that harbors three related, but genetically distinct, nitrogenases. Which one of these enzymes is operational at any particular time depends on which metal ions are present (1). For more than 50 years, the requirement of molybdenum for nitrogenase was thought to be mandatory (2), even though vanadium was demonstrated early on to be almost as stimulatory as Mo to the growth of some bacteria on N 2 as the sole nitrogen source (3). Despite these and other early experiments, the essentiality of Mo became entrenched. However, it is now clear that Mo is not essential for biological nitrogen fixation. Progress through the 1980's clearly identi...

α-Hydroxy coordination of mononuclear vanadyl citrate, malate and S-citramalate with N-heterocycle ligand, implying a new protonation pathway of iron–vanadium cofactor in nitrogenase

“…Here several accurate structural methods for small molecules converge in the determination of the proton in the α–hydroxy group of hydroxycarboxylates. Although there is a large gap between the coordination chemistry of mononuclear α–hydroxycarboxylato vanadium/molybdenum with the biological homocitrate system, and the vanadyl hydroxycarboxylates of citrate, malate or S -citramalate does not involve R -homocitrate, the present formation of unsymmetric mononuclear hydroxycarboxylato vanadyl compounds 1 ~ 6 could still be related to the coordination of homocitrato FeV-cofactor in nitrogenase [5, 49], that may show an unusual neutral α-hydroxy coordination in low oxidation state (+3 ~ +4) [50] as shown in Scheme 2.…”

Structural Basis of Biological Nitrile Reduction

“…The crystal structure of wild-type QueF complexed with substrate was determined using the molecular replacement Bayesian protocol in the program Phaser (16) and a search model generated from the structure of V. cholerae QueF (Protein Data Bank (PDB) ID 3BP1 (11)) by replacing nonhomologous residues with alanines. A solution with highest likelihood gain was obtained in the P3 2 21 space group. The partial model obtained in Phaser and describing the asymmetric unit was used for automatic tracing and refinement in the program Arp/Warp (17), which produced a significantly more complete model with a crystallographic R-factor of 0.35.…”

Nitrogenase reduction of carbon-containing compounds

“…It was later shown that hydrogen cyanide (HCN) is the substrate, while CN − is the inhibitor [59]. The 2 e − /H + , 4 e − /H + , and 6 e − /H + reduction of HCN was observed with the production of methyleneimine (CH 2 =NH), methylamine (CH 3 NH 2 ), methane (CH 4 ), and ammonia (NH 3 ) as products for both the Mo- and V-nitrogenases [60,61]. The hydrolysis of unstable methyleneimine led to the production of formaldehyde and ammonia as the final products (Table 1).…”

“…The hydrolysis of unstable methyleneimine led to the production of formaldehyde and ammonia as the final products (Table 1). The further reduction of formaldehyde to methanol was not found [61]. However, very small amounts of C2 products, C 2 H 4 and C 2 H 6 , were observed, which might be derived from an insertion mechanism accounting for the isocyanide reduction and coupling [59,62].…”