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 Fe(II)Fe(III) 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 the label is at the methyl carbon atom or when the methyl group is deuterated. These results suggest that acetate is oriented in the active site with its carboxylate group pointing toward the iron center, confirming a previous interpretation of X-ray crystallographic experiments. At the MMOH diiron core, four coordination positions that are not usually occupied by endogenous protein ligands are identified which can simultaneously accommodate DMSO, methanol, and water. The implications of these results for the catalytic mechanism of the enzyme are discussed.
ASJC Scopus subject areas
- Colloid and Surface Chemistry