Mn(III) species formed by the multi-copper oxidase MnxG investigated by electron paramagnetic resonance spectroscopy
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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 5B1g 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.
KeywordsParallel-mode EPR Multi-copper oxidase MnxG Mnx protein complex Mn(II) oxidation Zero-field splitting
The work was supported by the National Science Foundation Award Numbers CHE-1213699, CHE-1665455 to RDB, EAR-1231322 to WHC, CHE-1410688 to BMT, and CHE-1410353 to TGS. The EPR spectrometers at the CalEPR facility used in this study were funded by the National Institutes of Health (S10-RR021075) and the NSF (CHE-1048671).
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