Redox Properties of the Copper Centers in Particulate Methane Monooxygenase

Abstract Particulate methane monooxygenase (pMMO) and its homolog ammonia monooxygenase (AMO), central enzymes in the global carbon and nitrogen cycles, contain three highly conserved mononuclear copper centers, denoted CuB, CuC, and CuD. While these sites have been well characterized structurally and spectroscopically, with recent studies identifying CuD as the active site, their reduction potentials have not been measured. This knowledge gap has hindered efforts to determine how the two electrons and two protons required for substrate oxidation are sourced and delivered. Here, we have elucidated the redox properties of each copper center in pMMO/AMO by protein film voltammetry using samples with copper occupancies known from cryoelectron microscopy (cryoEM) structures. The reduction potential of the CuB site, −72 to −139 mV, is significantly lower than the ∼400 mV measured for the CuD site, with consistent values determined for pMMOs and AMO from different species. Mutation of a residue in the second sphere of CuB, Glu35, alters the redox potential and impairs cell growth and pMMO activity, indicating that redox chemistry at CuB might be functionally relevant. Finally, a Pourbaix diagram constructed for the CuBII/I and CuDII/I half-reactions suggests that a proton-coupled electron transfer (PCET) process occurs at the CuD site but not at the CuB site. Thermochemical analysis of this proposed PCET process is compatible with electron delivery by a quinol reductant. These results provide new context for future investigations of the pMMO and AMO reaction mechanisms.