Ca²⁺ effects on manganese oxidation by the bacterial multicopper oxidase complex Mnx

Dormant spores of manganese-oxidizing Bacillus sp. oxidize dissolved manganese, producing insoluble manganese oxide mineral. Many of them do so with the help of Mnx-a protein complex of a catalytically active multicopper oxidase enzyme MnxG bound to accessory proteins MnxE and MnxF-in a two-step reaction process, first oxidizing Mn(II) to Mn(III), then converting Mn(III) to the nanoparticulate MnO₂ product. In this report, the effect of Ca²⁺ ions on manganese biomineralization by Mnx from Bacillus sp. PL-12 is investigated by spectroscopic kinetic assays. The sigmoidal shape of the enzymatic progress curves is unaffected by Ca²⁺, indicating that the enzymatic mechanism is preserved. The rate of the overall reaction increases up to ∼10 μM of Ca²⁺, and then decreases. The Mn(III) trapping agent, pyrophosphate, was used to investigate the effect of Ca²⁺ on the two individual reaction steps. The acceleration/deceleration sequence with ∼10 μM Ca²⁺ optimum occurs during the conversion of Mn(III) to Mn(IV). The Mn(III)/Mn(IV) turnover rate shows a cooperative dependence on the Ca²⁺ concentration, indicating that two Ca²⁺ act simultaneously. The oxidation to the final Mn(IV) product is known to proceed via disproportionation of a Mn(III)(μ-OH)₂Mn(III) intermediate. We propose that the resulting Mn(IV)(μ-O)₂Mn(IV) dimers assemble tetramers via additional oxo bridge formation, a process promoted by Ca²⁺. Maturation of the tetramers via further polymerization is likewise promoted by Ca²⁺. A stoichiometry of 0.5Ca/Mn is seen for the Ca-induced shift in the Mn(III)/Mn(IV) equilibrium, indicating the binding of 1 Ca for every 2 Mn in the Mnx-produced MnO₂ nanoparticles.