Bacterial oxidation of Mn(II) is an important component of the Mn cycle in aquatic and terrestrial systems. Two classes of Mn(II) oxidases have been described: animal heme peroxidases and multicopper oxidases (MCOs). However, their biochemical mechanisms have not been well- characterized. Our group has investigated the Mnx complex from Bacillus sp., strain PL-12. Mnx is comprised of MnxG, an MCO, and two accessory proteins, MnxE and MnxF. MnxG is unusual for an MCO because it requires accessory proteins for maximum expression and activity and it catalyzes a two electron oxidation of a metal substrate . Using a variety of spectroscopic and microscopic techniques, we have gained important insights into the structure of Mnx and have begun to unravel how Mnx catalyzes two energetically distinct oxidation reactions, where substrate, intermediate, and product require different ligand coordination. We use native mass spectrometry coupled with electron microscopy to develop a structural model of Mnx with a diameter on the order of 8 nm, a molecular mass of 211 kDa, and a stoichiometry of MnxGE3F3. Surface induced dissociation (SID) shows that MnxE primarily binds one Cu with relatively high affinity; MnxF binds up to 3 Cu with lower affinity. Using various spectroscopic methods, we show that Mnx exploits manganese polynuclear chemistry to lower the Mn(III/II) reduction potential, enable oxidation of Mn(II) to Mn(III) and subsequently Mn(III) to Mn(IV), likely producing an oxo-bridged Mn(IV) dimer from which MnO2 nanoparticles nucleate. High resolution electron microscopy suggests that Mn oxide particles are released from the Mnx complex as clusters containing a few Mn and oxygen atoms, and that these clusters then aggregate into larger minerals. Our work shows the complexity of the novel biochemical mechanism by which Mnx forms Mn oxides. The structural studies also serve as the foundation for engineering such enzymes for biotechnological applications.

Mechanistic investigations of manganese(IV) oxide biomineralization catalyzed by a multicopper oxidase complex

255th National Meeting (2018)

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