Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

Abstract

Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios.

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

“…A chemical model was generated for the electrochemical oxidation of the enamine leading to dimerization at the C2a atom, suggesting significant electron spin density at this atom. Subsequent detailed work on PFOR clearly showed that the spin density is delocalized into the thiazolium ring but there indeed is a significant fraction at the C2a atom [85].…”

“…Early evidence for the existence of a C2a-hydroxyethylideneThDP radical and a free-radical mechanism on ThDP enzymes was obtained on pyruvate-ferredoxin oxidoreductase (PFOR) [82][83][84][85], an enzyme that converts pyruvate to acetylCoA in anaerobes. In addition to ThDP, the enzyme has three Fe 4 S 4 clusters forming a 40-50 Å long electron transfer chain.…”

One-carbon chemistry of oxalate oxidoreductase captured by X-ray crystallography

“…One explanation for how CoA binding to PFOR accelerates the second electron transfer step is that the additional negative charge brought to the active site by the deprotonated CoA thiol drives down the reduction potential of the HE-TPP radical, allowing the electron to move into the lowpotential [4Fe-4S] clusters (21). Whether this happens before or after nucleophilic attack by CoA, however, is still a matter of investigation (27).…”

“…This intermediate is analogous to the proposed state of the HE-TPP adduct in PFOR (SI Appendix, Fig. S10) (23,27). Such neutral species are thought to have a high barrier for electron transfer (34).…”

“…Taking into account what is known about TPP reaction intermediates in PFOR (SI Appendix, Fig. S10) (10,27) as well as our structural snapshots of OOR, we can propose a mechanism for OOR catalysis (Fig. 6).…”