Reactions of Nitric Oxide with the Reduced Non-Heme Diiron Center of the Soluble Methane Monooxygenase Hydroxylase

Abstract

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (H_red) were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mössbauer spectroscopy. Reaction of NO with H_red affords a dinitrosyl species, designated H_dinitrosyl, with optical spectra (λ_max = 450 and 620 nm) and Mössbauer parameters (δ = 0.72 mm/s, ΔE_Q = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The H_dinitrosyl species models features of the H_peroxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, H_dinitrosyl builds up with approximately the same rate constant as H_peroxo (∼26 s^-1) at 4 °C. In the absence of protein B, the kinetics of H_dinitrosyl formation were best fit with a biphasic A → B → C model, indicating the presence of an intermediate species between H_red and H_dinitrosyl. This result contrasts with the reaction of H_red with dioxygen, in which the H_peroxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.