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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.

Original publication

DOI

10.1021/ja076468k

Type

Journal article

Journal

J Am Chem Soc

Publication Date

06/02/2008

Volume

130

Pages

1662 - 1668

Keywords

Catalysis, Citric Acid Cycle, Electron Spin Resonance Spectroscopy, Free Radicals, Geobacillus stearothermophilus, Models, Molecular, Molecular Structure, Oxygen, Peroxides, Thiamine