Identification of the Valence and Coordination Environment of the Particulate Methane Monooxygenase Copper Centers by Advanced EPR Characterization

Abstract

Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol in methanotrophic bacteria. As a copper-containing enzyme, pMMO has been investigated extensively by electron paramagnetic resonance (EPR) spectroscopy, but the presence of multiple copper centers has precluded correlation of EPR signals with the crystallographically identified monocopper and dicopper centers. A soluble recombinant fragment of the pmoB subunit of pMMO, spmoB, like pMMO itself, contains two distinct copper centers and exhibits methane oxidation activity. The spmoB protein, spmoB variants designed to disrupt one or the other or both copper centers, as well as native pMMO have been investigated by EPR, ENDOR, and ESEEM spectroscopies in combination with metal content analysis. The data are remarkably similar for spmoB and pMMO, validating the use of spmoB as a model system. The results indicate that one EPR-active Cu(II) ion is present per pMMO and that it is associated with the active-site dicopper center in the form of a valence localized Cu(I)Cu(II) pair; the Cu(II), however, is scrambled between the two locations within the dicopper site. The monocopper site observed in the crystal structures of pMMO can be assigned as Cu(I). 14N ENDOR and ESEEM data are most consistent with one of these dicopper-site signals involving coordination of the Cu(II) ion by residues His137 and His139, the other with Cu(II) coordinated by His33 and the N-terminal amino group. 1H ENDOR measurements indicate there is no aqua (HxO) ligand bound to the Cu(II), either terminally or as a bridge to Cu(I).

New Trends in Oxidative Functionalization of Carbon–Hydrogen Bonds: A Review

“…For example, dinuclear copper complex 11 [221,224] and hexairon complex 15 (for the structure, see Figure 9) affording the products of cyclohexane oxidation in a very high yield of 46% (see Figure 10 [226]) can be considered as "inorganic models of methane monooxygenases." These enzymes contain dinuclear copper [317,318] …”

“…For example, dinuclear copper complex 11 [221,224] and hexairon complex 15 (for the structure, see Figure 9) affording the products of cyclohexane oxidation in a very high yield of 46% (see Figure 10 [226]) can be considered as "inorganic models of methane monooxygenases." These enzymes contain dinuclear copper [317,318] and iron [319] reaction centers, respectively. A new method of synthesis of heterometallic coordination compounds by spontaneous self-assembly (SSA) gave complexes which are very efficient catalysts in oxidations with peroxides (turnover frequency TOF was up to 11200 h´1 = 3.1 s´1) [160].…”

Methanobactin and the Link between Copper and Bacterial Methane Oxidation

“…The pMMO, in contrast, is a copper-and possibly iron-containing, membrane-associated enzyme that is associated with unusual intracytoplasmic membranes that take the form of vesicular disks in type I methanotrophs and paired peripheral layers in type II organisms (65)(66)(67)(68)(69)(70)(71)(72)(73)(74)(75). Intracytoplasmic membranes are enriched in pMMO and can be physically separated from the cytoplasmic membrane on the basis of sedimentation velocity in sucrose density gradients (76).…”

Elaboration of copper–oxygen mediated C–H activation chemistry in consideration of future fuel and feedstock generation

“…X-ray crystallographic studies exhibit that pMMO possesses three different metal active sites; dicopper, monocopper, and a zinc center [21]. Biochemical studies implicate an asymmetrical dinuclear copper active site [22-24], although Chan et al [25] hold the view that an as yet unseen pMMO trinuclear Cu center constitutes the active site. The dicopper site displays a short Cu–Cu distance, where the ligands are provided by two His's for one copper and the bidentate N-terminal histidine (‘His-brace’, as in LPMOs) for the other (Figure 1c).…”