Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption Spectroscopy

Abstract

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-Å resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO_iso), chemically reduced (pMMO_red), and chemically oxidized (pMMO_ox) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO_iso contains both Cu^I and Cu^II and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu−Cu interaction in all redox forms of the enzyme. The Cu−Cu distance increases from 2.51 to 2.65 Å upon reduction, concomitant with an increase in the average Cu−O/N bond lengths. Appropriate Cu₂ model complexes were used to refine and validate the EXAFS fitting protocols for pMMO_iso. Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe^III, is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.