Pulsed EPR Studies of Particulate Methane Monooxygenase from Methylococcus Capsulatus (Bath):  Evidence for Histidine Ligation

Particulate methane monooxygenase (pMMO), a membrane-bound metalloenzyme found in all obligate methanotrophs, catalyzes the 2-electron, dioxygen-dependent oxidation of methane to methanol. We have previously proposed that the catalytic site of pMMO is a copper cluster on the basis on the observed dependence of cellular growth and catalysis with copper ion concentration. The number of copper ions strongly bound by pMMO is large, up to 15 copper ions per pMMO unit, and we have suggested that they are primarily arranged in multinuclear clusters that are unlike other multinuclear copper centers found in metallobiochemistry. We recently presented a model that differentiated the pMMO copper content into subsets of copper that are associated with catalysis (C-clusters), on the basis of their susceptibility to oxidation by air from the Cu(I) to the Cu(II) state, and those copper ions that can only be oxidized by ferricyanide (E-clusters). Electron spin--echo modulation spectroscopy (ESEEM) has been a useful tool for the identification and characterization of nitrogenous ligands in copper(II)-containing metalloproteins. In this paper, we present our initial characterization of the copper(II) ions of oxidized pMMO from Methylococcus capsulatus (Bath) by ESEEM spectroscopy.