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

Methyldiazene (HN=N-CH₃) isotopomers labeled with ¹⁵N at the terminal or internal nitrogens or with ¹³C or ²H were used as substrates for the nitrogenase α-195^Gln- substituted MoFe protein. Freeze quenching under turnover traps an S = 1/2 state that has been characterized by EPR and ¹H-, ¹⁵N-, and ¹³C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NH_x] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N₂ reduction, H atoms sequentially add to the distal N before N-N bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NH_x] 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 [-NH_x] 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 [-NH_x] 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 α-195^Gln-substituted MoFe protein.