Unraveling the Mechanism of Manganese (II) Oxidation by Pseudomonas Putida

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The oxidation of soluble manganese(II) to insoluble Mn(III) and Mn(IV) oxyhydroxides has a profound effect on the environment. The highly reactive Mn oxide mineral phases scavenge numerous heavy metals, oxidize various toxic organic and inorganic compounds, and serve as electron acceptors for growth of anaerobic bacteria. In nature, most Mn oxides form through the activities of microorganisms, primarily bacteria. Studies of three phylogenetically-diverse Mn(II)-oxidizing bacteria show that a homologous gene encoding a multicopper oxidase-like protein is required for Mn(II) oxidation. The direct link between those genes and enzymatic oxidation of Mn(II), however, has yet to be established. This project focuses on the mechanism, regulation and function of Mn(II) oxidation in Pseudomonas putida strain GB-1. Spectroscopic evidence indicates that Mn(II) oxidation proceeds via two sequential one-electron steps, both of which are enzymatically catalyzed. The recent discovery of a second multicopper oxidase gene required for Mn(II) oxidation in GB-1, in addition to the previously described cumA, suggests that two multicopper (Mn) oxidases drive this catalytic process. The new 5.9 Kb gene resembles the putative Mn oxidase gene, mnxG, from Bacillus SG-1 in its highly conserved copper binding sites. This project will determine whether each protein catalyzes one step of the two-electron oxidation, or alternatively, whether they work in concert to catalyze the overall reaction. Mutagenesis studies point to other factors that mediate Mn(II) oxidation, including interactions with c-type cytochromes, protein transport and location of the native enzymes. Physiological studies indicate that substrate-ligand binding and induction/ inhibition of oxidation by other metals, specifically Fe, play significant roles in Mn(II) oxidation. The results of this research will provide new insights into the reasons why bacteria oxidize manganese(II), broaden our understanding of biogeochemical cycles and the natural attenuation of toxic metals and organic compounds.Broader Impacts: The results from this project may lead to improved technologies for environmental remediation. Additionally, it will contribute to the education of undergraduate and highly-qualified high school students through independent research projects and mentorships; and to a training program for middle school teachers, highlighting the connections between the chemistry, biology and geology of the oceans.