The Catalytic Copper Clusters of the Particulate Methane Monooxygenase from Methanotrophic Bacteria: Electron Paramagnetic Resonance Spectral Simulations

Abstract The particulate methane monooxygenase (pMMO) from Methylococcus capsulatus contains 14–15 reduced copper ions, and it has been proposed that these copper ions are arranged in the form of trinuclear clusters. Two of these clusters have been referred to as catalytic clusters (C‐clusters) and have been implicated in dioxygen activation and alkane hydroxylation. The remaining copper ions appear to provide a “reservoir” of reducing equivalents to replenish the electrons at the C‐clusters following dioxygen activation at the C‐clusters during turnover. Accordingly, they are normally reduced and have been called electron‐transfer clusters (E‐clusters). For pMMO prepared in highly enriched membranes, or purified as the pMMO‐detergent complex from these membranes, only the C‐cluster copper ions are oxidized. Recently, the low temperature EPR spectrum of as‐isolated pMMO was deconvoluted into a type 2 Cu(II) signal and a broad, but nearly isotropic EPR signal centered at g ˜ 2.1. Earlier magnetization and magnetic susceptibility measurements have suggested that the latter EPR signal, which is not sensitive to microwave power saturation, might arise from a ferromagnetically exchange‐coupled trinuclear Cu(II) cluster with J ≈ 20 cm −1 and D ≤ 0.05 cm −1 . Toward confirming these results, several triangular model Cu(II) complexes, both antiferromagnetically and ferromagnetically coupled and with well‐defined structural and ligand information, were reviewed to gain insights into magneto‐structural correlations. Spectral simulations of the pMMO cluster EPR signals were then performed based on the structural and spectroscopic information provided by the ferromagnetic model complexes. We show that only Cu(II) ions with proper g‐tensors and appropriate relative orientations between them can give rise to the unique EPR signal observed for the C‐cluster(s) in pMMO. The putative C‐cluster EPR signal observed for the as‐isolated pMMO at 3 K was best fitted by a triad of ferromagnetically coupled Cu(II) ions with the following sets of g‐values: (1.983, 2.030, 2.218), (1.983, 2.029, 2.218) and (2.000, 2.033, 2.207); and zero‐field splitting parameters D = 0.017 ± 0.002 cm −1 (175 ± 25 Gauss) and E/D = 0.15. The fit was not sensitive to the value of J so long as the exchange interaction was much larger than the Zeeman interaction (J » gβH).

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