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

可溶性锰（II）氧化成不溶性锰（III）和锰（IV）羟基氧化物对环境具有深远的影响。高反应性的锰氧化物矿物相吸收大量的重金属，氧化各种有毒的有机和无机化合物，并作为电子受体的厌氧细菌的生长。在自然界中，大多数锰氧化物通过微生物，主要是细菌的活动形成。三种遗传多样的锰（II）氧化细菌的研究表明，一个同源基因编码的多铜氧化酶样蛋白是必需的锰（II）氧化。然而，这些基因与Mn（II）的酶促氧化之间的直接联系尚未建立。本项目主要研究恶臭假单胞菌GB-1中Mn（II）氧化的机制、调控和功能。光谱证据表明，Mn（II）氧化通过两个连续的单电子步骤，这两个步骤都是酶催化的。最近发现的第二个多铜氧化酶基因所需的Mn（II）氧化GB-1，除了先前描述的cumA，表明两个多铜（Mn）氧化酶驱动这个催化过程。新的5.9 Kb基因类似于推定的锰氧化酶基因，mnxG，从芽孢杆菌SG-1在其高度保守的铜结合位点。该项目将确定每个蛋白质是否催化双电子氧化的一个步骤，或者它们是否协同工作以催化整个反应。诱变研究指出其他因素介导锰（II）氧化，包括与C型细胞色素，蛋白质运输和本地酶的位置的相互作用。生理学研究表明，底物-配体结合和诱导/抑制氧化的其他金属，特别是铁，发挥显着的作用，锰（II）氧化。这项研究的结果将为细菌氧化锰（II）的原因提供新的见解，拓宽我们对生物地球化学循环以及有毒金属和有机化合物的自然衰减的理解。更广泛的影响：该项目的结果可能会导致环境修复技术的改进。此外，它将通过独立的研究项目和指导，为本科生和高素质高中生的教育做出贡献;并为中学教师的培训计划做出贡献，强调海洋化学，生物学和地质学之间的联系。

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.