Copper Centers in the Cryo-EM Structure of Particulate Methane Monooxygenase Reveal the Catalytic Machinery of Methane Oxidation

Abstract

The particulate methane monooxygenase (pMMO) is the first enzyme in the C1 metabolic pathway in methanotrophic bacteria. As this enzyme converts methane into methanol efficiently near room temperature, it has become the paradigm for developing an understanding of this difficult C1 chemistry. pMMO is a membranebound protein with three subunits (PmoB, PmoA, and PmoC) and 12− 14 coppers distributed among different sites. X-ray crystal structures that have revealed only three mononuclear coppers at three sites have neither disclosed the location of the active site nor the catalytic mechanism of the enzyme. Here we report a cyro-EM structure of holo-pMMO from Methylococcus capsulatus (Bath) at 2.5 Å, and develop quantitative electrostatic-potential profiling to scrutinize the nonprotein densities for signatures of the copper cofactors. Our results confirm a mononuclear Cu I at the A site, resolve two Cu I s at the B site, and uncover additional Cu I clusters at the PmoA/PmoC interface within the membrane (D site) and in the water-exposed C-terminal subdomain of the PmoB (E clusters). These findings complete the minimal set of copper factors required for catalytic turnover of pMMO, offering a glimpse of the catalytic machinery for methane oxidation according to the chemical principles underlying the mechanism proposed earlier.

Coordination of the Copper Centers in Particulate Methane Monooxygenase: Comparison between Methanotrophs and Characterization of the CuCSite by EPR and ENDOR Spectroscopies

“…This identification of the coordination sphere of Cu B in both types of pMMO, combined with other work identifying Cu B as a monocopper site, ,, is incompatible with a recent cryoelectron microscopy structure of M. capsulatus (Bath) pMMO …”

“…This identification of the coordination sphere of Cu B in both types of pMMO, combined with other work identifying Cu B as a monocopper site, 14,15,17 is incompatible with a recent cryoelectron microscopy structure of M. capsulatus (Bath) pMMO. 38 Stochastic CW 1 H ENDOR of the Cu B Site. To refine our picture of the electronic and molecular structure of the Cu B site, we employed 35 GHz field-modulated stochastic CW 1 H ENDOR on whole cell Mc.…”

Unexpected Complexity of the Ammonia Monooxygenase in Archaea

“…Although there is debate on whether AmoC harbors the primary active site in AMO, there is clear evidence that the metal site in PmoC plays a critical role in the complex of methanotrophs 27,30,31 . While the archaeal AmoC lacks a substantial section found in all bacteria that corresponds to two transmembrane helices (Supplementary Fig.…”

“…This holds true for most of the diverse enzymes of the CuMMO (copper-dependent membrane monooxygenase) protein family, with a few notable exceptions. Crystal structures [22][23][24][25][26] and one cryo-EM structure 27 of particulate methane monooxygenase (pMMO) from five methanotrophs have consistently confirmed a threepolypeptide protomer (subunits-A, -B and -C) arranged in a trimer of α3β3γ3 configuration with at least two conserved metal sites in each protomer. Even so, the elucidation of the active site has remained ambiguous.…”

“…Even so, the elucidation of the active site has remained ambiguous. It was first proposed to reside in the PmoB subunit of pMMO 28 as recently supported by cryo-EM analysis 27 , while differing amino acid conservation in Verrucomicrobia 29 , a recent spectroscopic analysis 30 , and mutagenesis of a hydrocarbon monooxygenase 31 suggest its localization in the PmoC subunit.…”