Nitrogen Fixation Special Feature: A methyldiazene (HNNCH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism

摘要

Methyldiazene (HNNCH3) 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 = ½ 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 [-NHx] 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 [-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 α-195^Gln-substituted MoFe protein.