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. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

Fig. 1

EPR spectra (X-band) of the radical formed upon addition of pyruvate to PFORs from diverse organisms. A Moorella thermoaceticum [48], B Desulfovobrio africanus [39], C Halobacterium salinarium [10], D Methanosarcina barkeri [61].

Fig. 2

Ribbon representation of the homodimer of PFOR from D. africanus (PDB ID: 1KEK). The Fe-S clusters, divalent cations (green), and TPP-pyruvate adduct are highlighted as space filling models.

Fig. 3

EPR spectrum (77 K) of the HE-TPP radical obtained from samples made up from [2-¹³C]-pyruvate with PFOR from M. thermoacetica. The experimental spectrum was collected at high signal-to-noise ratio by scan averaging and subjected to resolution enhancement using Fourier methods [47]. The calculated spectrum was generated using the g values and hyperfine tensors, and line shape parameters described previously [48].

Fig. 4

Isosurface plots of spin density in an energy-minimized model of the HE-TPP radical obtained from DFT calculations (B3LYP/6-31G*//B3LYP/TZVP using Gaussian 09) [49] that include the aminopyrimidine moiety of TPP and the active site residues Arg 114 and Glu 64. The initial positions of atoms in the model were taken from PDB ID: 1KEK. The plot was created using the program Molekel 4.3 [62]. ¹³C hyperfine splitting tensors arising from the model were [8,7,21] (G) for ¹³C2α (experimental [6,6,18] (G)) and [5,4,3] (G) for ¹³C2 (experimental [4,4,9] (G)). These values are approximately midway between extremes calculated for the neutral and cationic HE-TPP radicals [48].

Scheme 1

Examples of bonds cleaved in TPP-dependent reactions

Scheme 2

Formation of lactyl-TPP

Scheme 3

Decarboxylation of lactyl-TPP to give HE-TPP

Scheme 4

One-electron oxidation of HE-TPP to produce the HE-TPP radical

Scheme 5

Overall reaction of PFOR

Scheme 6

Proposed model of a σ/n cation radical for the HE-TPPP radical

Scheme 7

Hypothetical CoA anion radical adducts

Scheme 8

Hypothetical radical combination mechanism

Scheme 9

Proposed mechanism of OOR