Reduction of cyclic and acyclic diazene derivatives by Azotobacter vinelandii nitrogenase: diazirine and trans-dimethyldiazene

Nitrogenase reduces N2 to NH3, but the mechanistic details are unclear. Diazene (N2H2), a proposed 2e(-)/2H(+) intermediate on the reduction pathway, is labile under typical enzyme assay conditions, and no firm evidence is available on whether or not it can be reduced by or inhibit nitrogenase. In this paper, we compare the interactions of Azotobacter vinelandii (Av) nitrogenase with two diazene analogues: diazirine, a photolabile diazene containing the azo (-N=N-) group in a strained, three-membered ring, and trans-dimethyldiazene, a diazene containing an unstrained trans-disubstituted N=N bond. Diazirine is reduced by nitrogenase under specific conditions to methane, methylamine, and ammonia in a ratio of ca. 1:2:4-5 with a Km value for all three products similar (0.05-0.09 mM) to that of dinitrogen (0.06-0.12 mM). The Km value of diazirine does not depend on the ratio of nitrogenase Fe protein (Av2) to nitrogenase MoFe protein (Av1) at Av2:Av1 ratios of 0.71 and 14.9. Diazirine potently and competitively inhibits acetylene reduction by Av nitrogenase with Ki = 0.03 mM and is predicted to inhibit H2 evolution completely at pressures >> Km. The experimental Henry's Law constant (1.50 M/atm) determined for trans-dimethyldiazene in H2O shows that it has about 20-fold higher solubility than diazirine in water at 30 degrees C. trans-Dimethyldiazene is reduced by nitrogenase under specific conditions to ammonia, methane and methylamine in a ratio of ca. 1:1:1 with Km values for the three products of 0.51-0.58 M. The product ratio does not change significantly when the component ratio (Av2:Av1) is varied over 2.06-13.62. trans-Dimethyldiazene reduction is inhibited non-competitively by CO and C2H2 with Ki values of ca. 0.0008 and 0.006 atm, respectively. The results are discussed with respect to the stereoelectronic differences between the two azo substrates. A "random-edge" reduction is compared with alternative schemes for the diazirine reduction. For trans-dimethyldiazene, initial C-N cleavage is proposed to yield CH4 and a bound CH3N2H species, which is then reduced to CH3NH2 and NH3.