Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate

Abstract

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 Ͻ1% of succinyl-CoA under physiological conditions. Significance: This work presents the novel discovery that the 5-carboxyl group affects enzymatic reactivity of 2-oxoglutarate.

Progress in the experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…Interestingly, on both E1o-ec and E1o-h, the same thiaminenamine-derived radical was observed by EPR spectroscopy, as were the reduced species derived from O 2 , superoxide anion radical and thence hydrogen peroxide [89]. The complicated fate of the enamine on E1o components is shown in Schemes 2 and 4.…”

“…7 [79,89]. Given that the k max of the enamine derived from C2a-hydroxyalkyl thiazolium salts is expected near 290-295 nm [76], several steady state and time-resolved experiments were carried out to confirm the assignment.…”

“…More recent studies by Anand et al [75] concluded that in the absence of the E2p-ec component, but with an external oxidizing agent 2,6-dichloropheno-indophenol (DCPIP), the 2-acetylThDP is an intermediate on E1p-ec, but in the presence of both E2p-ec and E3-ec, there is no evidence for the intermediate, suggesting instead that the reductive acetylation is direct with no intermediate. Similarly, the presence of DCPIP in the reaction of E1o-ec with OG also creates the 2-electron oxidation product, 2-succinylThDP [79,89].…”

“…Panel A, time dependence of build-up of C2-(a-hydroxy)-c-carboxy-propylThDP intermediate. Panel B, time dependence of enamine formation on E1o-ec[79,89]. Panel C, steady state circular dichroism resulting from mixing of E1o-ec and 2-oxoglutarate.…”

Progress in understanding the molecular oxygen paradox – function of mitochondrial reactive oxygen species in cell signaling

“…However, this event actually occurs on the E1 subunit, which augmented O 2˙− /H 2 O 2 production by OGDH (Mailloux et al, 2016a,b). It should be noted here that OGDH produces O 2˙− /H 2 O 2 from the E1 and E3 subunits; the former via thiamine radical formation and latter through its FAD prosthetic group (Nemeria et al, 2014). Thus, the GSH-mediated S-glutathionylation of the E1 subunit likely results in thiamine radical accumulation augmenting ROS production (Figure 3).…”