The long-term objective of this project is to understand the catalytic mechanism of acetyl-CoA synthase/carbon monoxide dehydrogenase (ACS/CODH), one of the most complex metalloenzymes known. This oxygen-sensitive bifunctional enzyme contains two nickel-iron-sulfur cluster active-sites which are unique in biology. They are connected by a protein tunnel through which CO migrates from one site to the other, controlled by a conformational change which is intricately correlated to the catalytic mechanism. One of the Ni ions may be reduced to a zero-valent state during reductive activation, and this ion may bind methyl and acetyl groups during catalysis;thus the enzyme catalyzes an organometallic reaction mechanism which is unique in biology. Indeed two specific aims for the next four years are to obtain direct physical evidence for the zero-valent state and for the Ni-bound methyl and acetyl adducts. Another aim is to determine the factors that control the conformation of the protein and to understand how protein conformation is choreographed with catalysis. For reasons that are poorly understood, the enzyme is heterogenous in that only ~ 30% of ACS/CODH molecules in a population are catalytically functional.
A fourth aim i s to determine the origin of this heterogeneity and eliminate it if possible. A cadre of spectrocopic methods will be used, including EPR and M""""""""ssbauer spectroscopy, NMR, X-ray absorption, and fluoresence. Stopped-flow kinetics and site-directed mutagenesis will also be used.

Public Health Relevance

Clostridium difficile, which contains this enzyme ACS/CODH, causes antibiotic-associated colitis, toxic megacolon, intestinal perforations and even death in humans. Our mechanistic study of ACS/CODH will help define the metabolic roles played by the enzyme in this pathogen, and identify strategies for preventing the proliferation of C. difficile in intestines. Also, ACS/CODH is important in environmental health, as it removes CO from the atmosphere and degrades TNT from abandoned military sites. ACS/CODH is involved in C1 metabolism and it contains a sophisticated tunnel through which CO migrates, impacting the field of metabolic channeling. A number of other metalloenzymes are heterogeneous in terms of catalytic function, and the studies described here might contribute to elucidating the reasons for such """"""""half-sites"""""""" reactivity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM046441-17
Application #
7687538
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1993-04-01
Project End
2012-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
17
Fiscal Year
2009
Total Cost
$305,055
Indirect Cost
Name
Texas A&M University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
Chakrabarti, Mrinmoy; Barlas, Mirza Nofil; McCormick, Sean P et al. (2015) Kinetics of iron import into developing mouse organs determined by a pup-swapping method. J Biol Chem 290:520-8
Chakrabarti, Mrinmoy; Cockrell, Allison L; Park, Jinkyu et al. (2015) Speciation of iron in mouse liver during development, iron deficiency, IRP2 deletion and inflammatory hepatitis. Metallomics 7:93-101
Schilter, David; Rauchfuss, Thomas B; Stein, Matthias (2012) Connecting [NiFe]- and [FeFe]-hydrogenases: mixed-valence nickel-iron dithiolates with rotated structures. Inorg Chem 51:8931-41
Xie, Wei; Parker, Janet L; Heaps, Cristine L (2012) Effect of exercise training on nitric oxide and superoxide/H?O? signaling pathways in collateral-dependent porcine coronary arterioles. J Appl Physiol (1985) 112:1546-55
Schilter, David; Rauchfuss, Thomas B (2012) Nickel-iron dithiolates related to the deactivated [NiFe]-hydrogenases. Dalton Trans 41:13324-9
Lindahl, Paul A (2012) Metal-metal bonds in biology. J Inorg Biochem 106:172-8
Schilter, David; Nilges, Mark J; Chakrabarti, Mrinmoy et al. (2012) Mixed-valence nickel-iron dithiolate models of the [NiFe]-hydrogenase active site. Inorg Chem 51:2338-48
Kamat, Siddhesh S; Bagaria, Ashima; Kumaran, Desigan et al. (2011) Catalytic mechanism and three-dimensional structure of adenine deaminase. Biochemistry 50:1917-27
Hess, Jennifer L; Hsieh, Chung-Hung; Brothers, Scott M et al. (2011) Self-assembly of dinitrosyl iron units into imidazolate-edge-bridged molecular squares: characterization including Mossbauer spectroscopy. J Am Chem Soc 133:20426-34
Kamat, Siddhesh S; Holmes-Hampton, Gregory P; Bagaria, Ashima et al. (2011) The catalase activity of diiron adenine deaminase. Protein Sci 20:2080-94

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