Refining details of the structural and electronic properties of the CuB site in pMMO enzyme through sequential molecular dynamics/CPKS-EPR calculations

Abstract

This work investigated the structural and electronic properties of the copper mononuclear site of the PmoB part of the pMMO enzyme at the molecular level. We propose that the Cu_B catalytic site in the soluble portion of pMMO at room temperature and under physiological conditions is a mononuclear copper complex in a distorted octahedral arrangement with the residues His³³, His¹³⁷, and His¹³⁹ on the equatorial base and two water molecules on the axial axis. Our view was based on the molecular dynamics results and DFT calculations of the electronic paramagnetic resonance parameters and comparisons with experimental EPR data. This new proposed model for the Cu_B site brings additional support concerning the recent experimental evidence, which pointed out that a saturated coordination sphere of the copper ion in the Cu_B center is an essential factor that makes it less efficient than the Cu_C site in the methane oxidation. Therefore, according to the Cu_B site model proposed here, an additional step involving a displacement of at least one water molecule of the copper coordination sphere by the O₂ molecule prior to its activation must be necessary. This scenario is less likely to occur in the Cu_C center once this one is buried in the alpha-helices, which are part of the pMMO structure bound to the membrane wall, and consequently located in a less solvent-exposed region. In addition, we also present a simple and efficient sequential S-MD/CPKS protocol to compute EPR parameters that can, in principle, be expanded for the study of other copper-containing proteins.