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

可溶性锰(II)氧化成难溶的氢氧化锰(III)和氢氧化锰(IV)对环境有深远的影响。高活性的氧化锰矿物相清除大量的重金属，氧化各种有毒的有机和无机化合物，并作为厌氧细菌生长的电子受体。在自然界中，大多数锰氧化物是通过微生物，主要是细菌的活动形成的。对三种系统发育不同的锰(II)氧化细菌的研究表明，锰(II)氧化需要一个编码多铜氧化酶样蛋白的同源基因。然而，这些基因与酶促氧化锰(II)之间的直接联系尚未确定。本课题主要研究恶臭假单胞菌GB-1中Mn(II)氧化的机理、调控及其功能。光谱证据表明，Mn(II)的氧化通过两个连续的单电子步骤进行，这两个步骤都是在酶的催化下进行的。最近在GB-1中发现了除先前描述的CuMA外，第二个多铜(II)氧化所需的多铜氧化酶基因，表明这一催化过程是由两个多铜(Mn)氧化酶驱动的。新的5.9kb基因在高度保守的铜结合位点上与推测的来自芽孢杆菌SG-1的锰氧化酶基因mnxG相似。该项目将确定每种蛋白质是否催化两电子氧化中的一步，或者它们是否协同工作来催化整个反应。诱变研究指出了其他介导Mn(II)氧化的因素，包括与C型细胞色素的相互作用、蛋白质运输和天然酶的位置。生理学研究表明，底物-配体结合和其他金属，特别是铁对氧化的诱导/抑制在锰(II)氧化中起着重要作用。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和有毒金属和有机化合物的自然衰减的理解。广泛的影响：该项目的结果可能导致环境修复技术的改进。此外，它还将通过独立的研究项目和指导，促进本科生和高素质高中生的教育；并促进中学教师的培训计划，突出海洋化学、生物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.