Bioinorganic Chemistry of Carbon Monoxide Dehydrogenase

描述（由申请人提供）：本项目的目的是阐明CO脱氢酶（a.k.a.乙酰辅酶A合酶- ACS），强调其中所含的独特Ni-Fe-S簇的结构和功能。该酶是一种α-2-β-2四聚体，催化两个反应：CO2可逆还原为CO和合成乙酰辅酶A。前者发生在β亚基中的一个Ni-Fe-S簇，而后者发生在α亚基中的另一个Ni-Fe-S位点。CO迁移通过的隧道连接这两个站点。反应机制是“有机金属”的性质;如何镍促进这一含水蛋白质基质？发生在两个网站的机械步骤是同步的，使其成为一个有吸引力的系统，研究代谢通道和活性位点耦合的细节。ACS在生物技术上是有用的;含有它的生物体可以降低大气中的CO水平并降解TNT。ACS是在艰难梭菌中发现的，艰难梭菌是一种致病微生物，每年造成约2000人死亡。考虑到这种酶的复杂性，本项目中使用的一般策略是分别研究每种活性，然后将观察到的特性与双功能酶的特性进行比较;这种方法可能会揭示通道和催化偶联的一些复杂性。这种方法是可能的，因为可以制备能够催化乙酰辅酶A合成的分离的重组α亚基，以及能够催化CO/CO2氧化还原的β亚基的同系物。许多这些发展（重组生物合成的活性α，发现的隧道和活性位点耦合）产生的努力，导致在11个出版物的前一个授权期。用于该项目的方法包括酶动力学（停流，快速冷冻淬火和稳态），光谱学（EPR，穆斯堡尔和XAS）和X射线衍射。一个快速冷冻淬火仪器，使样品在可靠的厌氧条件下制备已经建成，应该是对现有技术的改进。动力学数据将被模拟，最终导致一个全面的机制模型描述酶的一般动力学行为。已经获得α晶体，表明可以实现X射线衍射结构。这些研究需要重复纯化至少5种不同的蛋白质;需要为制备型FPLC/HPLC提供资金。

DESCRIPTION (provided by applicant): The objective of this project is to elucidate the catalytic mechanism of CO dehydrogenase (a.k.a. acetyl-CoA synthase - ACS), emphasizing the structure and function of the unique Ni-Fe-S clusters contained therein. The enzyme is an alpha-2-beta-2 tetramer that catalyzes two reactions; the reversible reduction of CO2 to CO and the synthesis of acetyl-CoA. The former occurs at one Ni-Fe-S cluster in the beta subunit, while the latter reaction occurs at another Ni-Fe-S site in alpha. A tunnel through which CO migrates connects the two sites. The reaction mechanism is "organometallic" in nature; how does Ni promote this within an aqueous protein matrix? Mechanistic steps occurring at the two sites are synchronized, making it an attractive system to investigate details of metabolic channeling and active-site coupling. ACS is useful biotechnologically; organisms containing it reduce atmospheric levels of CO and degrade TNT. ACS is found in Clostridium difficile, a pathogenic organism responsible for the deaths of about 2000 people annually. Given the complexity of this enzyme, the general strategy used in this project will be to study each activity separately, and then compare observed properties with those of the bifunctional enzyme; such an approach may reveal some of the complexities of channeling and catalytic coupling. This approach is possible because isolated recombinant alpha subunits able to catalyze the synthesis of acetyl-CoA can be prepared, as can a homolog of the beta subunit that is able to catalyze CO/CO2 redox. Many of these developments (recombinant biosynthesis of active alpha, discovery of the tunnel and of active site coupling) arose from efforts of the previous granting period which resulted in 11 publications. The methodologies used for the project include enzyme kinetics (stopped-flow, rapid freeze-quench, and steady-state), spectroscopy (EPR, Mossbauer, and XAS), and X-ray diffraction. A rapid-freeze-quench instrument that allows samples to be prepared under reliably anaerobic conditions has been constructed, and should be an improvement over existing technology. Kinetic data will be simulated, ultimately leading to a comprehensive mechanistic model describing the general kinetic behavior of the enzyme. Crystals of alpha have been obtained, suggesting that an X-ray diffraction structure may be achievable. These studies require repetitive purification of at least 5 different proteins; funding for a preparative FPLC/HPLC is requested.