title: A methyldiazene (HN=N-CH3)-derived species bound to the nitrogenase active-site FeMo cofactor: Implications for mechanism.
abstract: 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 alpha-195Gln-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 [-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 alpha-195Gln-substituted MoFe protein.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA.
authors.forename: Brett M
authors.initials: BM
authors.lastname: Barney
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Lukoyanov
authors.forename: Tran-Chin
authors.initials: TC
authors.lastname: Yang
authors.forename: Dennis R
authors.initials: DR
authors.lastname: Dean
authors.forename: Brian M
authors.initials: BM
authors.lastname: Hoffman
authors.forename: Lance C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D007094:Imides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000378:metabolism*; D009584:Nitrogen / Q000737:chemistry; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D011522:Protons
chemical_list: D007094:Imides; D008983:Molybdoferredoxin; D011522:Protons; C038868:methyl diazene; D009584:Nitrogen