A methyldiazene (HN=WN-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor:: Implications for mechanism

Methylcliazene (HN=N-CH3) isotopomers labeled with N-15 at the terminal or internal nitrogens or with C-13 or H-2 were used as substrates for the nitrogenase alpha-195(Gln)-substituted MoFe protein. Freeze quenching under turnover traps an S = 1/2 state that has been characterized by EPR and H-1-, N-15-, and C-13-electron nuclear double resonance spectroscopies. These studies; disclosed the following: (i) a methyidiazene-derived species is bound to the activesite FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methylcliazene terminal N; and (iii) the internal N from methylcliazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methylcliazene. In the Chatt-Schrock mechanism for N-2 reduction, H atoms sequentially add to the distal N before WN bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to N-N bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before N-N cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in alpha-195(Gln)-substituted MoFe protein.