Critical evaluation of three cryo-EM structures of particulate methane monooxygenase by quantum refinement.

Abstract

Particulate methane monooxygenase (pMMO) is an enzyme that converts methane into methanol at ambient temperature and pressure. Over the past three decades, the metal content and location of the active site have been highly controversial. Recent single-particle cryogenic electron-microscopy (cryo-EM) structures have furthered this debate. In this study, three cryo-EM structures (PDB entries 7s4h, 7s4j and 7ev9) are analysed by quantum refinement (QR). This approach augments traditional structural refinement with quantum-mechanical (QM) calculations for a small but interesting part of the protein (in this case, the copper sites). Our results indicate that the bis-His (CuA) site is correctly modelled as a mononuclear copper site in all three structures. The His-brace (CuB) site is also best modelled as mononuclear in all structures, although it was suggested to be a binuclear site in PDB entry 7ev9. The CuC site, which is observed only in PDB entry 7s4j, is correctly modelled and is probably reduced in the structure. The CuD putative active site, observed only in PDB entry 7s4h, is also mononuclear, but a water molecule might at least intermittently coordinate to the copper ion. On the other hand, our study does not find any support for the five additional copper ions suggested to be present in PDB entry 7ev9, including the suggested trinuclear active site and two sites in the so-called copper sponge. Instead, more chemically reasonable structures and better fit to both the cryo-EM and QM data are obtained if these copper ions are replaced with water molecules. This study illustrates the potential of QR as a standard component of cryo-EM studies for metal sites, for which reliable empirical restraints are missing.