Enzymology of the reductive acetyl-CoA pathway

描述(申请人提供)：本研究申请集中于Wood-LJungdahl途径中三个关键酶的机制研究：CO脱氢酶/乙酰辅酶A合成酶(CODH/ACS)、甲基转移酶(METR)和类皮质铁-硫蛋白(CFeSP)。对该体系的研究丰富了微生物学、生物化学和矿物生物化学领域，揭示了大分子通道和未知金属团簇的结构。这些研究为这些蛋白质如何利用生物有机金属中间体催化基团转移反应以及C-C和C-S键的形成和裂解提供了洞察力，使有机体能够吸收二氧化碳和有毒气体CO。这个系统是理解生物无机化学原理、复杂的蛋白质-蛋白质相互作用以及蛋白质如何协调氧化还原反应与化学催化的范例。这一应用的重点是对三类主要的镍-CODH进行多学科的结构-功能研究：单功能的CODH，产乙酸菌的双功能的CODH/ACS，以及来自产乙酸菌的双功能的CODH/ACS(也称为乙酰辅酶A脱羧酶合成酶，ACDs)。由于这些酶发挥着不同的生理作用，因此它们表现出相应不同的催化性能，包括不同的底物沟道机制，CO氧化/二氧化碳还原的催化偏向，以及电化学性质。包括酸碱催化剂(S)在内的质子转移网络，在所有CODH类中都是保守的，将被进一步研究。CODH机制中的早期中间体(S)将被捕获和表征。将对ACS机理中的内部电子转移反应进行表征。我们将进一步调查CODH/ACS中的CO通道如何测量CO的供应和需求，并调查CODH/ACS中的CODH和ACS站点是如何协调的。我们还将确定CFeSP-Metr络合物的晶体结构，并研究Metr与CFeSP反应中主要构象变化所起的作用。对PFOR的研究将检验动力学耦合假说，包括鉴定CoA与羟乙基-TPP自由基反应形成的一种拟议的自由基物种。与公共健康相关：我们正在研究厌氧微生物吸收二氧化碳和一氧化碳的途径中的关键酶。这些研究包括阐明金属离子在生物学中的新作用(金属-碳键、新的异金属簇合物和亲核金属离子)，表征新的底物衍生的自由基中间体，以及描述酶内气体底物的通道。

DESCRIPTION (provided by applicant): This research application focuses on mechanistic studies of three key enzymes in the Wood-Ljungdahl pathway: CO dehydrogenase/acetyl-CoA synthase (CODH/ACS), methyltransferase (MeTr), and the corrinoid iron-sulfur protein (CFeSP). Studies on this system have enriched the areas of microbiology, biochemistry, and metallobiochemistry in revealing the structures of macromolecular channels and previously unknown metal clusters. These studies are providing insight into how these proteins use bioorganometallic intermediates in catalyzing group transfer reactions and C-C and C-S bond formation and cleavage allowing organisms to assimilate carbon dioxide and the toxic gas, CO. This system serves as a paradigm for understanding bioinorganic chemical principles, complex protein-protein interactions, and how proteins coordinate redox reactions with chemical catalysis. The major focus of this application is on multidisciplinary structure-functions studies of the three major classes of Ni-CODHs: the monofunctional CODH, the bifunctional CODH/ACS from acetogenic bacteria, and the bifunctional CODH/ACS (also called acetyl-CoA decarbonylase synthase, ACDS) from aceticlastic methanogens. Because these enzymes play different physiological roles, they are hypothesized to exhibit correspondingly disparate catalytic properties, including different mechanisms of substrate channeling, catalytic biases for CO oxidation/carbon dioxide reduction, and electrochemical properties. The proton transfer network including the acid-base catalyst(s), conserved among all CODH classes, will be further investigated. The early intermediate(s) in the CODH mechanism will be trapped and characterized. The internal electron transfer reaction in the ACS mechanism will be characterized. We will further investigate how the CO channel in CODH/ACS meters the supply of and demand for CO and investigate how the CODH and ACS sites in CODH/ACS are coordinated. We also will determine the crystal structure of the CFeSP- MeTr complex and investigate the proposed role of a major conformational change in the reaction of MeTr with the CFeSP. Studies on PFOR will test a kinetic coupling hypothesis, including identification of a proposed radical species formed by reaction of CoA with the hydroxyethyl-TPP radical. PUBLIC HEALTH RELEVANCE: We are studying the key enzymes in a pathway by which anaerobic microbes assimilate carbon dioxide and carbon monoxide. These studies involve the elucidating new roles of metal ions in biology (metal-carbon bonds, new heterometallic clusters, and nucleophilic metal ions), characterizing novel substrate-derived radical intermediates, and describing channels for gaseous substrates within an enzyme.