title: Off-pathway, oxygen-dependent thiamine radical in the Krebs cycle.
abstract: The catalytic cofactor thiamine diphosphate is found in many enzymes of central metabolism and is essential in all extant forms of life. We demonstrate the presence of an oxygen-dependent free radical in the thiamine diphosphate-dependent Escherichia coli 2-oxoglutarate dehydrogenase, which is a key component of the tricarboxylic acid (Krebs) cycle. The radical was sufficiently long-lived to be trapped by freezing in liquid nitrogen, and its electronic structure was investigated by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR). Taken together, the spectroscopic results revealed a delocalized pi radical on the enamine-thiazolium intermediate within the enzyme active site. The radical is generated as an intermediate during substrate turnover by a side reaction with molecular oxygen, resulting in the continuous production of reactive oxygen species under aerobic conditions. This off-pathway reaction may account for metabolic dysfunction associated with several neurodegenerative diseases. The possibility that the on-pathway reaction may proceed via a radical mechanism is discussed.
authors.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK.
authors.forename: René A W
authors.initials: RA
authors.lastname: Frank
authors.forename: Christopher W M
authors.initials: CW
authors.lastname: Kay
authors.forename: Judy
authors.initials: J
authors.lastname: Hirst
authors.forename: Ben F
authors.initials: BF
authors.lastname: Luisi
mesh_terms: D002384:Catalysis; D002952:Citric Acid Cycle; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D008958:Models, Molecular; D015394:Molecular Structure; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism*; D010545:Peroxides / Q000378:metabolism; D013831:Thiamine / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D010545:Peroxides; D010100:Oxygen; D013831:Thiamine