Abstract
Particulate methane monooxygenase (pMMO) is a copper-dependent integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus (Mcc.) capsulatus (Bath) and Methylomicrobium (Mm.) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure, as observed by electron paramagnetic resonance (EPR) spectroscopy. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z reveals a mononuclear copper center in the PmoB subunit and indicates that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment and provide valuable insight into pMMO function.
Highlights
Of essential copper ions are not lost during isolation from the membranes
The crystal structure of 20Z-pMMO provides some insight into how removal from the membrane could affect activity
PmoB only contains two transmembrane helices, and PmoA is sandwiched between PmoB and PmoC, features that may contribute to their structural stability in detergent micelles
Summary
To systematically investigate loss of pMMO activity, methane oxidation activity was measured for as-isolated, solubilized, purified, and bicelle-reconstituted pMMO samples. pMMO activity assays are typically performed using either NADH or duroquinol as a reductant. Methane oxidation activity was measured for as-isolated membranes, solubilized and purified pMMO in detergent (DDM), and bicelle (3% (w/v) DMPCCHAPSO) reconstituted pMMO using both reductants (Fig. 1). Significantly lower than the NADH-driven activity for BathpMMO and not detected for 20Z-pMMO (Fig. 1) For both pMMOs, reconstitution into bicelles recovers the methane oxidation activity of solubilized and purified samples using duroquinol as a reductant (Fig. 1 and Table S1). It may be that an NDH-2 or other components of the electron transport chain responsible for NADH-dependent methane oxidation are not properly reassembled after solubilization and reconstitution of 20Z-pMMO For both pMMOs, duroquinol-driven activity is significantly higher for bicelle-reconstituted samples than for asisolated membranes and is comparable with NADH-driven activity in membranes (Fig. 1 and Table S1). Solubilized or purified pMMO samples were reconstituted in bicelles without the addition of copper, suggesting that bicelles alone are responsible for the recovered activity
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