UC Davis

The multi-copper oxidase (MCO) MnxG from marine Bacillus bacteria plays an essential role in geochemical cycling of manganese by oxidizing Mn²⁺(aq) to form manganese oxide minerals at rates that are three to five orders of magnitude faster than abiotic rates. The MCO MnxG protein is isolated as part of a multi-protein complex, denoted as Mnx, which includes one MnxG unit and a hexamer of MnxE₃F₃ subunit. During the oxidation of Mn²⁺(aq) catalyzed by the Mnx protein complex, an enzyme-bound Mn(III) species was trapped recently in the presence of pyrophosphate (PP) and analyzed using parallel-mode electron paramagnetic resonance (EPR) spectroscopy. Herein, we provide a full analysis of this enzyme-bound Mn(III) intermediate via temperature dependence studies and spectral simulations. This Mnx-bound Mn(III) species is characterized by a hyperfine-coupling value of A(⁵⁵Mn) = 4.2 mT (corresponding to 120 MHz) and a negative zero-field splitting (ZFS) value of D = - 2.0 cm^-1. These magnetic properties suggest that the Mnx-bound Mn(III) species could be either six-coordinate with a ⁵B_1g ground state or square-pyramidal five-coordinate with a ⁵B₁ ground state. In addition, as a control, Mn(III)PP is also analyzed by parallel-mode EPR spectroscopy. It exhibits distinctly different magnetic properties with a hyperfine-coupling value of A(⁵⁵Mn) = 4.8 mT (corresponding to 140 MHz) and a negative ZFS value of D = - 2.5 cm^-1. The different ZFS values suggest differences in ligand environment of Mnx-bound Mn(III) and aqueous Mn(III)PP species. These studies provide further insights into the mechanism of biological Mn²⁺(aq) oxidation.