Copper Binding Sites in the Manganese-Oxidizing Mnx Protein Complex Investigated by Electron Paramagnetic Resonance...

Manganese-oxide minerals (MnO_x) are widely distributed over the Earth's surface, and their geochemical cycling is globally important. A multicopper oxidase (MCO) MnxG protein from marine Bacillus bacteria plays an essential role in producing MnO_x minerals by oxidizing Mn²⁺(aq) at rates that are 3 to 5 orders of magnitude faster than abiotic rates. The MnxG protein is isolated as part of a multiprotein complex denoted as "Mnx" that includes accessory protein subunits MnxE and MnxF, with an estimated stoichiometry of MnxE₃F₃G and corresponding molecular weight of ≈211 kDa. Herein, we report successful expression and isolation of the MCO MnxG protein without the E₃F₃ hexamer. This isolated MnxG shows activity for Mn²⁺(aq) oxidation to form manganese oxides. The complement of paramagnetic Cu(II) ions in the Mnx protein complex was examined by electron paramagnetic resonance (EPR) spectroscopy. Two distinct classes of type 2 Cu sites were detected. One class of Cu(II) site (denoted as T2Cu-A), located in the MnxG subunit, is identified by the magnetic parameters g_∥ = 2.320 and A_∥ = 510 MHz. The other class of Cu(II) sites (denoted as T2Cu-B) is characterized by g_∥ = 2.210 and A_∥ = 615 MHz and resides in the putative hexameric MnxE₃F₃ subunit. These different magnetic properties correlate with the differences in the reduction potentials of the respective Cu(II) centers. These studies provide new insights into the molecular mechanism of manganese biomineralization.