Bioinorganic Chemistry of Carbon Monoxide Dehydrogenase

描述(申请人提供)：本项目的目标是阐明一氧化碳脱氢酶的催化机理。乙酰辅酶A合成酶)，强调其中包含的独特的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：二氧化碳可逆还原为CO和乙酰辅酶A的合成。前者发生在β亚基中的一个Ni-Fe-S簇上，后者发生在α亚基中的另一个Ni-Fe-S中心。CO迁移所通过的隧道连接这两个站点。反应机制本质上是“有机金属”的；镍是如何在水蛋白质基质中促进这一作用的？在这两个部位发生的机械性步骤是同步的，这使得它成为一个有吸引力的系统来研究代谢通道和活性部位耦合的细节。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。在艰难梭菌中发现了ACS，这是一种致病微生物，每年导致约2000人死亡。考虑到这种酶的复杂性，这个项目中使用的一般策略将是分别研究每种活性，然后将观察到的性质与双功能酶的性质进行比较；这样的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。其中许多进展(活性α的重组生物合成、隧道的发现和活性部位偶联的发现)源于前一个授权期的努力，导致了11篇论文的发表。该项目使用的方法包括酶动力学(停流、快速冷冻-淬火和稳态)、光谱分析(EPR、穆斯堡尔和XAS)和X射线衍射。一种可以在可靠的厌氧条件下制备样品的快速冷冻急冷仪已经建成，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理模型描述酶的一般动力学行为。已经获得了α的晶体，这表明X射线衍射结构是可能实现的。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.