Background: The human 2-oxoglutarate dehydrogenase complex, comprising E1o, E2o, and E3 components, catalyzes conversion of 2-oxoglutarate to succinyl-CoA. Results: Human E1o generates both a thiamin-enamine-derived radical and the reactive oxygen species, superoxide, and hydrogen peroxide. Conclusion: Human E1o produces reactive oxygen species at a rate of <1% of succinyl-CoA under physiological conditions. Significance: This work presents the novel discovery that the 5-carboxyl group affects enzymatic reactivity of 2-oxoglutarate. Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2-hydroxy)--carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2-hydroxy)--carboxypropylidene thiamin diphosphate (the ThDP-enamine/C2-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O-2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an off-pathway side reaction comprising less than 1% of the on-pathway reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.