EPR Spectroscopic and Computational Characterization of the Hydroxyethylidene-Thiamine Pyrophosphate Radical Intermediate of Pyruvate:Ferredoxin Oxidoreductase

Abstract

The radical intermediate of pyruvate:ferredoxin oxidoreductase (PFOR) from Moorella thermoacetica was characterized using electron paramagnetic resonance (EPR) spectroscopy at Xband and D-band microwave frequencies. EPR spectra obtained with various combinations of isotopically labeled substrate (pyruvate) and coenzyme (thiamine pyrophosphate (TPP)), were analyzed by spectral simulations. Parameters obtained from the simulations were compared with those predicted from electronic structure calculations on various radical structures. The g-values and 14 N/ 15 N-hyperfine splittings obtained from the spectra are consistent with a planar, hydroxyethylidene-thiamine pyrophosphate (HE-TPP) π-radical, in which spin is delocalized onto the thiazolium sulfur and nitrogen atoms. The 1 H-hyperfine splittings from the methyl group of pyruvate and the 13 C-hyperfine splittings from C2 of both pyruvate and TPP are consistent with a model in which the pyruvate-derived oxygen atom of the HE-TPP radical forms a hydrogen bond. The hyperfine splitting constants and g-values are not compatible with those predicted for a nonplanar, σ/n-type cation radical.Pyruvate:ferredoxin oxidoreductase (E.C.1.2.7.1) (PFOR 1 ) is a member of the 2-keto acid oxidoreductase family. This enzyme plays a central role in anaerobic fermentative metabolism by catalyzing the reversible, CoA-dependent, oxidative decarboxylation of pyruvate to acetylCoA and CO 2 (3). The two electrons generated in the oxidative reaction are transferred through [4Fe-4S] clusters in PFOR to an oxidized ferredoxin (4). PFOR is found in microbes lacking mitochondria from all three kingdoms (5). PFOR enzymes from different species have been grouped into three types depending on subunit composition (6). The different types of PFOR enzymes have between one and three [4Fe-4S] clusters. clusters are electron transfer cofactors serving as way-stops for electrons exiting the active site in the forward † This work was supported by grants from the National Institutes of Health: GM35752 G.H.R.; GM39451 S.W.R.; DK44083 T.P.B.; and Center Grant 1P20RR17675 to the University of Nebraska. S.O.M was supported by an NIH Predoctoral Training Grant T32 GM 08293. *To whom correspondence should be addressed. Telephone: (608) 262-0509; Fax: (608) 265-2904; Email: reed@biochem.wisc.edu. ‖ Current address: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066 1 Abbreviations: PFOR, pyruvate:ferredoxin oxidoreductase; CoA, coenzyme A; TPP, thiamine pyrophosphate; HE-TPP, hydroxyethylidene-thiamine pyrophosphate; EPR, electron paramagnetic resonance; ENDOR, electron nuclear double resonance; CW continuous wave; DFT, density functional theory; S/N, signal-to-noise ratio; RMSD, root mean square deviation; POX, pyruvate oxidase. to an acetyl radical followed by radical recombination with a CoA-thiyl radical (11). Elucidation of the structure of the HE-TPP radical intermediate is essential to an eventual understanding of the subsequent steps in the overall reacti...

Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…The C2α-hydroxyethylideneThDP cation radical and the C2(C2α-hydroxy)-γ-carboxypropylidene cation radical-Early evidence for the existence of a C2α-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 40-50Å long electron transfer chain.…”

A CW and pulse EPR spectrometer operating at 122 and 244 GHz using a quasi-optical bridge and a cryogen-free 12 T superconducting magnet

“…To date only a few pulse HF-EPR (frequency higher than 95 GHz) spectrometers have been described in the literature. Smimov et al [6] and Gerfen et al [7] are using a 130 GHz continuous-wave (CW) and pulse EPR bridge developed by V. Krymov using IMPATT amplifiers and heterodyne quadrature detection. In the group of R. G. Griffin an instrument was developed for EPR, ENDOR, and DNP at 140 GHz [8,9]; Prisner et al [10] described a 180 GHz pulsed instrument, while Schmidt et al [11,12] developed a pulse EPR spectrometer operating at 270 GHz.…”

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin

“…If POX was to stabilize perfectly conjugated enamine and π-type radical intermediates, this would seemingly lead to an increase of the activation barriers for FAD reduction and nucleophilic attack of phosphate onto the ThDP radical. It is interesting to note in that context that the X-ray structure of the related ThDP radical in the ThDP enzyme pyruvate:ferredoxin oxidoreductase (PFOR) was not compatible with a planar ThDP radical (11), although EPR analysis suggested formation of a delocalized, π-type radical (12).…”