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.

Fig. 1.

A view of the FeMo cofactor highlighting Fe atoms 2, 3, 6, and 7 (numbering from Protein Data Bank ID code 1M1N), along with MoFe protein amino acids ligands (α-275^Cys and α-442^His) and the side chains of α-195^His and α-70^Val. The figure was generated by using the Protein Data Bank ID code 1M1N. Fe is green, S is yellow, Mo is purple, C is gray, O is red, and N is blue.

Scheme 1.

Methyldiazene synthetic scheme. Hydroxylamine-O-sulfonic acid (3) was synthesized as a white precipitate after reaction of chlorosulfonic acid (1) and hydroxylamine (2, where the N atom was either ¹⁵N or ¹⁴N). N-methylhydroxylamine (5) was synthesized by the reduction of nitromethane (4, where the N atom was either ¹⁴N or ¹⁵N and the C atom was either ¹²C or ¹³C). Methyldiazene (6) was prepared by the base-catalyzed reaction of hydroxylamine-O-sulfonic acid (3) with N-methylhydrolyamine (5), and the gas was isolated by freezing. See Materials and Methods for details.

Fig. 2.

Methyldiazene inhibition of proton reduction activity. The percentage of maximal H₂ evolution activity (nmol H₂/min per mg of MoFe protein) remaining in the presence of increasing methyldiazene is shown for the WT (△) and α-195^Gln (○) MoFe proteins. The maximum quantity of methyldiazene added to a 9-ml vial assuming 100% yield is shown.

Fig. 3.

EPR spectra are shown for the resting state (Top) and turnover trapped states with methylhydrazine (Middle) or methyldiazene (Bottom) present. EPR conditions are described in Materials and Methods. Calculated g values are noted for some inflections.

Fig. 4.

35-GHz ENDOR spectra. (A) ¹⁵N Mims ENDOR spectra of m(CH₃NNH). For H¹⁵NNCH₃, ν = 34.808 GHz, g = 2.03, π/2 = 52 ns, τ = 400 ns, T = 25.352 μs, radio frequency (RF) = 20 μs, eight scans, 30 shots per point, 20-ms shot repetition time, 2 K. For HN¹⁵NCH₃, ν = 34.784 GHz, and other conditions are the same as for H¹⁵NNCH₃. Spectra are normalized for comparison. (B) As in A, but narrower scan and g = 2.01, τ = 552 ns, and 15 scans. Backgrounds have been corrected by simple subtractions as needed. (C) Continuous wave (CW) ¹H-ENDOR of m(CH₃NNH) in H₂O and D₂O. Conditions were: microwave frequency of 35.057–35.171 GHz, modulation amplitude = 4 G, RF sweep speed = 1 MHz/s, bandwidth of RF broadened to 100 kHz, 2 K. Methyldiazene is abbreviated as HNNMe.

Fig. 5.

Alternating and distal N₂ reduction mechanisms at a metal center (M). Essential intermediates are shown for N₂ reduction mechanisms with distal proton addition (d-mechanism) (Left) and alternating proton addition (a-mechanism) (Right), bound end-on to a metal center (M). Small straight arrows represent the addition of H⁺/e⁻ to substrate. Methyldiazene is placed where it might enter into each mechanism. Only the boxed intermediates are consistent with the ENDOR data for the methyldiazene-derived species trapped on the FeMo cofactor.