Preparation and characterization of a (Cu, Zn)-pMMO from Methylococcus capsulatus (Bath)

Abstract

We report the preparation of a (Cu, Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu, Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu, Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu, Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu, Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.