Use of a Coulombic Potential Framework for Interrogating the Copper Resolvability, Identity, and Nuclearity in pMMO Cryo‐ EM Maps

ABSTRACT

Particulate methane monooxygenase (pMMO) catalyzes the oxidation of methane to methanol under ambient conditions, yet the identity and nuclearity of its copper centers remain controversial. Interpretation of recent high‐resolution cryo‐electron microscopy (cryo‐EM) structures is complicated by the fact that cryo‐EM maps represent Coulombic electrostatic potentials rather than electron densities, making metal‐center assignment fundamentally different from X‐ray crystallography. Here, we develop a Coulombic potential map analysis framework for evaluating metal‐center nuclearity in cryo‐EM structures. The approach combines electrostatic‐potential simulation map based on updated Mott–Bethe scattering formalism to experimental map comparison and atom‐resolvability assessment using Q ‐scores. We applied this framework to the CuA and CuB copper centers of pMMO from Methylococcus capsulatus (Bath) across cryo‐EM structures spanning 2.14–2.48 Å resolution. Analysis of the CuA site, an undisputed monocopper center, demonstrates that the signal of a fully occupied copper ion is intrinsically modest and exhibits valence‐dependent features, with Cu(I) producing a broader potential distribution than Cu(II). Using CuA as a benchmark, we re‐examined CuB and found that the cryo‐EM maps support a mononuclear Cu(II) model in nanodisc‐reconstituted structures and a dinuclear Cu(I) model in the detergent‐isolated structure in a reduced state supporting NADH‐dependent catalytic turnover. These results establish a practical framework that is useful for evaluating metal centers in cryo‐EM maps to suggest that CuB nuclearity is strongly influenced by enzyme redox state, and readily extendable to other metalloenzymes.