Enzymology of the Reductive Acetyl-CoA Pathway

点击翻译按钮获取中文摘要

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.