Biochemical Mechanism of Mercury Methylation

项目摘要/摘要 汞甲基化的生化机制 我们的目标是确定汞(Hg)甲基化的生化机制，以及 在低氧的地下环境中发生的重要污染物转化。我们会 鉴定HGCA和HgcB，这两种蛋白质是汞甲基化所必需的 厌氧微生物。与橡树岭国家实验室(ORNL)汞合作 科学焦点领域(SFA)计划，我们生产的hgcb含有高水平的[4Fe- 4S]辅因子和HGCA的可溶钴胺(Cbl)结合区。最近的工作导致了 下列工作假设：(A)Hgcb中的两个铁-硫团簇从a 低电位氧化还原酶(丙酮酸铁氧还蛋白氧化还原酶、氢酶、一氧化碳 脱氢酶等)；(B)hgcB将这些还原当量转移到 HGCA，将其从Co(III)转化为超亲核Co(I)状态；(C)Cys73和C- 末端邻近半胱氨酸残基(脱硫弧菌ND132中的Cys95和Cys96) HgcB结合汞；和(D)HGCA催化甲基四氢叶酸(CH3-H4叶酸)依赖 Co(I)-Cbl甲基化生成甲基-Co(III)，然后甲基转移 甲基钴(III)转化为汞(II)产生甲基汞。我们的两个实验目标是：(1) 表征hgcB的结构和功能，并研究其作为汞载体和氧化还原的作用 HGCA和PFOR的合作伙伴和(2)表征相互作用和甲基化反应 涉及HcgB和HGCA。我们的实验将使用各种各样的生物物理和 生化技术使我们能够表征这些蛋白质的多维角色 在结合重金属、进行电子转移反应和催化甲基转移时， 最终产生一种强有力的有毒神经毒素。这些实验包括光谱学 (核磁共振、电子顺磁共振、共振拉曼等)、动力学(稳态和暂态)、电化学 和结合测量(表面等离子激元共振、等温量热、核磁共振等)。 我们的结果将揭示甲基汞产生的生化机制，揭示 对HGCA和HGCB催化的新型甲基转移反应有基本认识。 这项工作一般与重金属的微生物转化有关。这个 动力学参数将为代谢和反应转运模型提供关键输入 ORNL小组和其他人用来预测汞从单个生物体到生态系统的循环。 因此，我们的工作将有助于理解控制汞的命运和转化的过程 在水生环境中，这对于减少对人类和生态系统的风险是重要的 这些汞甲基化生物繁衍生息的地方。 。

Project Summary/Abstract Biochemical Mechanism of Mercury Methylation Our goal is to determine the biochemical mechanism of mercury (Hg) methylation, an important contaminant transformation that occurs in hypoxic subsurface environments. We will characterize HgcA and HgcB, the two proteins shown to be required for Hg methylation by anaerobic microorganisms. In collaboration with the Oak Ridge National Laboratory (ORNL) Hg Science Focus Area (SFA) program, we have produced HgcB containing high levels of its [4Fe- 4S] cofactor and a soluble cobalamin (Cbl) binding domain of HgcA. Recent work has led to the following working hypotheses: (a) the two iron-sulfur clusters in HgcB receive electrons from a low-potential oxidoreductase (pyruvate ferredoxin oxidoreductase, hydrogenase, CO dehydrogenase, etc.); (b) HgcB transfers these reducing equivalents to the Cbl cofactor of HgcA, converting it from Co(III) to the supernucleophilic Co(I) state; (c) Cys73 and the C- terminal vicinal cysteine residues (Cys95 and Cys96 in Desulfovibrio desulfuricans ND132) of HgcB bind Hg; and (d) HgcA catalyzes the methyltetrahydrofolate (CH3-H4folate)-dependent methylation of the Co(I)-Cbl to generate methyl-Co(III) followed by transfer of the methyl group of methyl-Co(III) to Hg(II) producing MeHg. Our two experimental objectives are to: (1) characterize the structure and function of HgcB and examine its roles as a Hg carrier and redox partner to HgcA and PFOR and (2) characterize the interactions and the methylation reactions involving HcgB and HgcA. Our experiments will use a wide variety of biophysical and biochemical techniques allowing us to characterize the multidimensional roles of these proteins in binding heavy metals, performing electron transfer reactions, and catalyzing methyl transfers, ultimately generating a potent and toxic neurotoxin. The experiments include spectroscopy (NMR, EPR, resonance Raman, etc.), kinetics (steady-state and transient), electrochemistry and binding measurements (surface plasmon resonance, isothermal calorimetry, NMR, etc). Our results will uncover the biochemical mechanism of MeHg production, revealing a fundamental understanding of the novel methyl transfer reactions catalyzed by HgcA and HgcB. This work will be generally relevant to the microbiological transformations of heavy metals. The kinetic parameters will provide key input for metabolic and reactive transport models that can be used by the ORNL group and others to predict Hg cycling from single organisms to ecosystems. Thus, our work will help understand the processes that control the fate and transformation of Hg in aquatic environments, which is important for mitigating risk to humans and ecosystems in which these Hg-methylating organisms thrive. .