Formation of a Novel Nickel-Iron Cluster and its Biological Role in Acetate Activation

产甲烷菌是产生甲烷气体作为其代谢的最终产物的微生物，它们对广泛的厌氧微生物分解过程至关重要。这些过程包括反刍动物和其他动物的消化，自然水生环境中的腐烂，以及用于城市和工业废物处理的人为过程。对我们的社会来说，甲烷一方面作为温室气体带来了潜在的问题，但同时它也是一种非常有用的清洁燃料。自然界中产生的近三分之二的甲烷来自产甲烷菌对醋酸盐的分解，该项目提供了这些生物如何在醋酸盐中进行碳-碳键切割的新分子细节。产醋酸和产甲烷微生物在厌氧条件下使用一种非常不寻常的酶促过程从单碳前体/产物中合成和分解大量乙酸，其中乙酸的碳-碳键被激活。在这一过程中，镍扮演着不可或缺的角色，作为一个独特的Ni-Fe/S簇的组成部分，位于一个5亚基多酶复合物（乙酰辅酶a脱碳酶/合成酶ACDS）的活性位点，该复合物负责Methanosarcina的醋酸裂解。Ni-Fe簇与配合物的β亚基结合，这也是底物CoA和乙酰辅酶a结合的位点。虽然现在很清楚Ni是碳-碳键激活的必要元素，但缺乏Ni- fe中心的结构信息，并且对Ni- fe簇的形成和组装所涉及的步骤知之甚少。这些信息对于清楚地了解Ni在醋酸盐活化中的作用至关重要。本项目的目标是通过表征辅助蛋白ACDS- orf在Ni插入含铁脱酶过程中的作用，确定ACDS β -亚基活性位点金属簇组装途径的步骤。镍的掺入是通过光谱方法和酶功能测定来监测的。参与β亚基Ni-Fe中心组装的其他蛋白质正在通过分子生物学双杂交测定和标准生化分离和分析方法的结合来寻找。镍如何在ACDS β亚基中作为Ni-Fe活性中心的一部分的详细知识将从实验中获得，以表征镍的配位环境。x射线吸收光谱方法（Ni EXAFS和Ni L-edge光谱）提供了Ni的几何形状、配体数量、化学性质和平均键距。位点定向诱变实验被用于鉴定作为Ni潜在配体的氨基酸残基，突变体将以其结合Ni的能力、经历配位环境的改变和醋酸活化的功能为特征。该项目的研究结果将为Ni-Fe/S簇的结构、功能和形成提供新的信息，从而更好地了解其生物化学及其在催化醋酸活化中的独特生理作用。

Methanogens are microorganisms that produce methane gas as the end product of their metabolism, and they are critical to a wide range of anaerobic microbiological decomposition processes. Such processes include digestion in ruminants and other animals, decay in natural aquatic environments, and man-made processes such as those used for municipal and industrial waste treatment. For our society, methane, on one-hand poses potential problems as a greenhouse gas, while at the same time it also serves as an extremely useful, clean-burning fuel. Nearly two-thirds of the methane produced in Nature is derived from decomposition of acetate by methanogens, and this project is contributing new molecular details of how these organisms carry out cleavage of the carbon-carbon bond in acetate. Acetogenic and methanogenic microorganisms carry out synthesis and breakdown of large quantities of acetic acid from one-carbon precursors/products under anaerobic conditions using a highly unusual enzymatic process in which the carbon-carbon bond of acetic acid is activated. Nickel plays an indispensable role in this process, serving as a component of a unique Ni-Fe/S cluster at the active site of a 5-subunit-containing multienzyme complex (designated ACDS for acetyl-CoA decarbonylase/synthase) responsible for acetate cleavage in species of Methanosarcina. The Ni-Fe cluster is bound to the beta subunit of the complex, which also is the site for binding of substrates CoA and acetyl-CoA. Although it is now clear that Ni is an essential element for carbon-carbon bond activation, structural information on the Ni-Fe center is lacking, and little is known about the steps involved in formation and assembly of the Ni-Fe cluster. Such information is critical to develop a clear understanding of how Ni functions in the activation of acetate. The goal of this project is to identify steps in the pathway of assembly of the ACDS beta-subunit active site metal cluster by characterizing the role of the accessory protein ACDS-ORF in the process of Ni insertion into the Fe-containing apoenzyme. Incorporation of nickel is monitored by spectroscopic methods and enzymatic functional assays. Additional proteins that participate in the assembly of the beta-subunit Ni-Fe center are being sought by a combination of molecular biological two-hybrid assays and standard biochemical methods of fractionation and analysis. Detailed knowledge of how nickel functions as part of the Ni-Fe active center in the ACDS beta subunit will be obtained from experiments to characterize the coordination environment of nickel. X-ray absorption spectroscopic methods (Ni EXAFS and Ni L-edge spectroscopy) are providing the geometry, number of ligands to Ni, their chemical nature, and average bond distances. Site-directed mutagenesis experiments are being used to identify amino acid residues serving as potential ligands to Ni, and mutants will be characterized for their ability to bind Ni, undergo changes in coordination environment and to function in acetate activation. The results from this project will provide new information on the structure, function and formation of an unusual Ni-Fe/S cluster, leading to a better understanding of its biochemistry and its unique physiological role in catalyzing the activation of acetate.