Small Molecule Binding to the Mixed-Valent Diiron Center of Methane Monooxygenase Hydroxylase from Methylococcus capsulatus (Bath) as Revealed by ENDOR Spectroscopy

Abstract

The binding of several exogenous molecules at the active site of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) is described. The interaction of methanol, the product of methane hydroxylation, with the mixed-valent FeIIFeIII form of the enzyme induces a change in the EPR spectrum of the unmodified protein from g = 1.940, 1.865, and 1.740 to one with g1 = 1.963 and g2 = 1.862. This spectral change arises from the coordination of methanol to the diiron center, as demonstrated by electron nuclear double resonance (ENDOR) spectroscopic studies of mixed-valent MMOH prepared with CD3OH added to a DMSO-treated sample, which displayed a 2H interaction with an isotropic hyperfine splitting of ∼0.5 MHz. A detailed analysis indicates that methanol binds to the ferrous iron without displacing a terminally bound water molecule identified previously. Acetate labeled with 13C at the carboxylate carbon atom also gives rise to ENDOR signals, whereas no such signals are seen when ...