Enzymology of the Reductive Acetyl-CoA Pathway

摘要 该提案重点关注Wood-Ljungdahl途径中关键酶的机制研究 CO/CO2固定和乙酰辅酶A合成。这些酶是CO脱氢酶（CODH）、乙酰辅酶A 合成酶（ACS）、类咕啉铁硫蛋白（CFeSP）及其同源甲基转移酶（MeTr），和 丙酮酸铁氧还蛋白氧化还原酶（PFOR）。对这一途径的研究将继续丰富 微生物学，生物化学和生物无机化学，揭示了重要的新见解， 大分子通道的结构，以前未知的金属簇，低价的不寻常的作用， 镍和钴离子作为亲核试剂，辅酶A的新生化作用，有机金属 复合物作为酶催化的中间体，以及大的构象运动， 活性位点与多个结合伴侣和氧化还原介体相互作用。我们将使用光谱， 诱变和动力学实验，以表征CODH和ACS的催化机制， 阐明连接CODH和ACS的CO通道中疏水口袋的性质 活性位点，并提供对构象变化的洞察，协调开放和关闭的 气体通道。对于ACS，我们最近开发了产生接近化学计量量的方法， 催化循环中的每种中间体，并计划确定它们的结构和电子性质。 在CFeSP和MeTr的研究中，我们计划阐明蛋白质-蛋白质复合物和构象 驱动甲基和电子转移的变化。在一个催化循环中，C-末端B12结合 CFeSP的CTD结构域必须与MeTr、CFeSP的小亚基和A簇相互作用 ACS的FeS结构域，以及当需要还原活化时CFeSP的FeS结构域。我们将 确定晶体结构并进行小角X射线散射实验 CFeSP与其结合伴侣之间的复合物。我们还将执行机械和结构 嵌合构建体的研究，其中CTD与其每种结合配偶体连接， 单独表达的域。在PFOR（及其CoA非依赖性同系物，草酸盐）的研究中， 氧化还原酶），我们计划表征CODH和PFOR之间的复合物及其在CO2中的作用 沟道效应和电子转移。我们还将阐明CoA在引起10万倍的 从底物诱导自由基中间体到完整铁-硫的电子转移速率的增加 集群

Abstract This proposal focuses on mechanistic studies of the key enzymes in the Wood-Ljungdahl pathway of CO/CO2 fixation and acetyl-CoA synthesis. These enzymes are CO dehydrogenase (CODH), acetyl-CoA synthase (ACS), the corrinoid iron-sulfur protein (CFeSP) and its cognate methyltransferase (MeTr), and pyruvate ferredoxin oxidoreductase (PFOR). Studies of this pathway will continue to enrich the areas of microbiology, biochemistry and bioinorganic chemistry by revealing significant new insights into the structures of macromolecular channels, previously unknown metal clusters, unusual roles of low-valent nickel and cobalt ions as nucleophiles, novel biochemical roles of Coenzyme A, organometallic complexes as intermediates in enzymatic catalysis, and large conformational movements that permit active sites to interface with multiple binding partners and redox mediators. We will use spectroscopic, mutagenesis and kinetic experiments to characterize the catalytic mechanisms of CODH and ACS, to elucidate the properties of hydrophobic pockets in the CO channel that connect the CODH and ACS active sites and provide insight into the conformational changes that coordinate opening and closing of the gas channel. For ACS, we have recently developed methods to generate nearly stoichiometric amounts of each intermediate in the catalytic cycle and plan to determine their structural and electronic properties. In studies of the CFeSP and MeTr, we plan to elucidate the protein-protein complexes and conformational changes that drive methyl and electron transfer. During one catalytic cycle, the C-terminal B12-binding domain (CTD) of the CFeSP must interface with MeTr, the small subunit of the CFeSP and the A-cluster of ACS, as well as the FeS domain of the CFeSP when reductive activation is required. We will determine the crystal structures and perform small angle X-ray scattering experiments of the key complexes between the CFeSP and its binding partners. We also will perform mechanistic and structural studies of chimeric constructs in which the CTD is linked to each of its binding partners and of these separately expressed domains. In studies of PFOR (and its CoA-independent homolog, oxalate oxidoreductase), we plan to characterize the complex between CODH and PFOR and its role in CO2 channeling and electron transfer. We also will elucidate the novel role of CoA in causing a 100,000-fold increase in rate of electron transfer from a substrate-induced radical intermediate to an integral iron-sulfur cluster.