This proposal focuses on structure-function studies on the key enzymes in the Wood-Ljungdahl pathway. Understanding the mechanisms of these enzymes is significant to the areas of enzymology, bioinorganic chemistry and microbiology. This pathway is important to human biochemistry since acetogens and methanogens are components of the intestinal flora. Studies of this pathway are uncovering the principles by which metal centers in enzymes perform nucleophilic reactions and couple redox reactions to chemical catalysis. The Clostridium thermoaceticum methyl transferase is the ideal paradigm for the class of B12-dependent methyl transferases. An important objective is to increase the resolution of the atomic model of methyl transferase and determine the structure with both B12 and CH3-H4folate bound. X-ray diffraction, site-directed mutagenesis, and kinetic studies will be used to identify the proton transfer pathway that leads to activation of CH3-H4folate for nucleophilic attack by the corrinoid iron-sulfur protein. Coordination chemistry of the cobalt center has been proposed to play a controlling role in redox chemistry and catalysis of B12-dependent methyl transferases. It is planned to measure the extent to which the lower axial ligand controls catalysis and redox chemistry and to determine the atomic structure of the corrinoid iron-sulfur protein. Mechanistic studies on CO dehydrogenase/acetyl-CoA synthase will include: determining its crystal structure; elucidating how the CO dehydrogenase and acetyl-CoA synthase active sites communicate to coordinate electron and carbon flow; determining whether the precursors of the carbonyl, methyl, and acetyl groups of acetyl-CoA bind to the Ni or Fe components of Cluster A in acetyl-CoA synthase; determining the binding order of methyl, carbonyl, and CoA moieties to Cluster A; and developing a way to express recombinant CO dehydrogenase/acetyl-CoA synthase in an active form. The electronic structure of a substrate-derived hydroxyethyl-TPP radical that has been shown to be a catalytic intermediate in the mechanism of the C. thermoaceticum pyruvate:ferredoxin oxidoreductase will be elucidated. The hypothesis that CO2 is channeled from pyruvate:ferredoxin oxidoreductase to CO dehydrogenase will be tested. Studies will be focused on determining how CoA binding affects electron transfer and catalysis and if a CoA radical may be a catalytic intermediate.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM039451-13A1
Application #
6131912
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Jones, Warren
Project Start
1991-08-01
Project End
2004-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
13
Fiscal Year
2000
Total Cost
$292,194
Indirect Cost
Name
University of Nebraska Lincoln
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
Chen, Percival Yang-Ting; Aman, Heather; Can, Mehmet et al. (2018) Binding site for coenzyme A revealed in the structure of pyruvate:ferredoxin oxidoreductase from Moorella thermoacetica. Proc Natl Acad Sci U S A 115:3846-3851
Gibson, Marcus I; Chen, Percival Yang-Ting; Johnson, Aileen C et al. (2016) One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography. Proc Natl Acad Sci U S A 113:320-5
Gibson, Marcus I; Brignole, Edward J; Pierce, Elizabeth et al. (2015) The Structure of an Oxalate Oxidoreductase Provides Insight into Microbial 2-Oxoacid Metabolism. Biochemistry 54:4112-20
Wang, Vincent C-C; Islam, Shams T A; Can, Mehmet et al. (2015) Investigations by Protein Film Electrochemistry of Alternative Reactions of Nickel-Containing Carbon Monoxide Dehydrogenase. J Phys Chem B 119:13690-7
Bachmeier, Andreas; Wang, Vincent C C; Woolerton, Thomas W et al. (2013) How light-harvesting semiconductors can alter the bias of reversible electrocatalysts in favor of H2 production and CO2 reduction. J Am Chem Soc 135:15026-32
Wang, Vincent C-C; Ragsdale, Stephen W; Armstrong, Fraser A (2013) Investigations of two bidirectional carbon monoxide dehydrogenases from Carboxydothermus hydrogenoformans by protein film electrochemistry. Chembiochem 14:1845-51
Wang, Vincent C-C; Can, Mehmet; Pierce, Elizabeth et al. (2013) A unified electrocatalytic description of the action of inhibitors of nickel carbon monoxide dehydrogenase. J Am Chem Soc 135:2198-206
Chaudhary, Yatendra S; Woolerton, Thomas W; Allen, Christopher S et al. (2012) Visible light-driven CO2 reduction by enzyme coupled CdS nanocrystals. Chem Commun (Camb) 48:58-60
Ando, Nozomi; Kung, Yan; Can, Mehmet et al. (2012) Transient B12-dependent methyltransferase complexes revealed by small-angle X-ray scattering. J Am Chem Soc 134:17945-54
Ragsdale, Stephen W; Yi, Li; Bender, G√ľne? et al. (2012) Redox, haem and CO in enzymatic catalysis and regulation. Biochem Soc Trans 40:501-7

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