Mechanistic enzymology of CO dehydrogenase from Clostridium thermoaceticum. Progress report, August 15, 1993--March 24, 1995
The final steps in acetyl-CoA biosynthesis by anaerobic bacteria are performed by carbon monoxide dehydrogenase (CODH), a nickel/iron-sulfur protein. An important achievement was to establish conditions under which acetyl-CoA synthesis by purified enzymes equals the in vivo rate of acetate synthesis. Under these optimized conditions we established that the rate limiting step in the synthesis of acetyl-CoA from methyl-H{sub 4}folate, CO and CoA is likely to be the methylation of CODH by the methylated corrinoid/iron-sulfur protein. We then focused on stopped flow studies of this rate limiting transmethylation reaction and established its mechanism. We have studied the carbonylation of CODH by infrared and resonance Raman spectroscopy and determined that the [Ni-Fe{sup 3-4}S{sub 4}]-CO species which has been characterized by magnetic resonance methods can be described as [Ni-X-Fe{sub 3-4}S{sub 4}]-C{equivalent_to}O. We showed that this species is the catalytically competent precursor of the carbonyl group of acetyl-CoA. We have made progress in the synthesis of seleno-coenzyme A, which we will use to probe the binding of CoA to CODH. We also have compared the CODH from Methanosarcina thermophila with the C. thermoaceticum enzyme by EPR and electrochemical methods and found that the metal sites of these enzymes are remarkably similar given the evolutionary separation between archaea and bacteria domains.
- Research Organization:
- Nebraska Univ., Lincoln, NE (United States)
- Sponsoring Organization:
- USDOE, Washington, DC (United States)
- DOE Contract Number:
- FG02-91ER20053
- OSTI ID:
- 10190626
- Report Number(s):
- DOE/ER/20053-1; ON: DE93002236
- Resource Relation:
- Other Information: PBD: [1992]
- Country of Publication:
- United States
- Language:
- English
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