REDUCTION OF CYCLIC AND ACYCLIC DIAZENE DERIVATIVES BY AZOTOBACTER-VINELANDII NITROGENASE - DIAZIRINE AND TRANS-DIMETHYLDIAZENE

Nitrogenase reduces N-2 to NH3, but the mechanistic details are unclea r. Diazene (N2H2), a proposed 2e(-)/2H(+) intermediate on the reductio n pathway, is labile under typical enzyme assay conditions, and no fir m evidence is available on whether or not it can be reduced by or inhi bit nitrogenase. In this paper, we compare the interactions of Azotoba cter 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 n itrogenase under specific conditions to methane, methylamine, and ammo nia in a ratio of ca. 1:2:4-5 with a K-m value for all three products similar (0.05-0.09 mM) to that of dinitrogen (0.06-0.12 mM). The K-m v alue of diazirine does not depend on the ratio of nitrogenase Fe prote in (Av2) to nitrogenase MoFe protein (Av1) at Av2:Av1 ratios of 0.71 a nd 14.9. Diazirine potently and competitively inhibits acetylene reduc tion by Av nitrogenase with K-i = 0.03 mM and is predicted to inhibit H-2 evolution completely at pressures much greater than K-m. The exper imental Henry's Law constant (1.50 M/atm) determined for trans-dimethy ldiazene 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 methy lamine in a ratio of ca. 1:1:1 with K-m values for the three products of 0.51-0.58 M. The product ratio does not change significantly when t he component ratio (Av2:Av1) is varied over 2.06-13.62. trans-Dimethyl diazene reduction is inhibited non-competitively by CO and C2H2 With K , values of ca. 0.0008 and 0.006 atm, respectively. The results are di scussed with respect to the stereoelectronic differences between the t wo azo substrates. A ''random-edge'' reduction is compared with altern ative schemes for the diazirine reduction. For trans-dimethyldiazene, initial C-N cleavage is proposed to yield CH4 and a bound CH3N2H speci es, which is then reduced to CH3NH2 and NH3.