Mechanism of the Clostridium thermoaceticum Pyruvate:Ferredoxin Oxidoreductase:  Evidence for the Common Catalytic Intermediacy of the Hydroxyethylthiamine Pyropyrosphate Radical

Abstract

The cofactor content and mechanism of pyruvate:ferredoxin oxidoreductase (PFOR) are controversial. By using rapid freeze-quench EPR and stopped-flow spectroscopy, the elementary steps that constitute the first half-reaction of the Clostridiumthermoaceticum PFOR mechanism were elucidated. A hydroxyethyl-TPP (HE-TPP) radical was identified and characterized as a transient intermediate, and for the first time, the kinetic competence of this substrate-derived radical was demonstrated. When the C. thermoaceticum PFOR was reacted with pyruvate and CoA, it had a lifetime of only approximately 100 ms. The results described here suggest that this radical intermediate is often not detected in studies of alpha-ketoacid oxidoreductases because it rapidly decays. It is postulated here that the HE-TPP radical is an intermediate in the mechanism of all PFORs irrespective of the number of 4Fe-4S clusters and will be detected in all PFORs when rapid mixing methods are used. The C. thermoaceticum PFOR was shown to contain two 4Fe-4S clusters, as concluded earlier [Wahl, R. C., & Orme-Johnson, W. H. (1987) J. Biol. Chem. 262, 10489-10496]. The first reductive half-reaction was shown to involve the following steps: (i) reaction with pyruvate with PFOR to form the hydroxyethylidene-TPP intermediate; (ii) one-electron transfer to reduce one of the two Fe4S4 clusters and yield the HE-TPP radical; and, (iii) reaction with CoA resulting in formation of acetyl-CoA, rapid decay of the HE-TPP radical intermediate, and reduction of the second Fe4S4 cluster. Thus, at the end of the first half-reaction, the two Fe4S4 clusters are fully reduced. The rate of the third step was found to depend on the CoA concentration (k = 35 per s at saturating concentrations of CoA); however, in its absence, this step was slower by approximately 4400-fold.

Radical Phosphate Transfer Mechanism for the Thiamin Diphosphate- and FAD-Dependent Pyruvate Oxidase from Lactobacillus plantarum. Kinetic Coupling of Intercofactor Electron Transfer with Phosphate Transfer to Acetyl-thiamin Diphosphate via a Transient FAD Semiquinone/Hydroxyethyl-ThDP Radical Pair

“…The net rate constant of the phosphorolytic cleavage of AcThDP (k′ 6 3 , and k′ 4 ). Because all of these rate constants have been already determined, the calculation of k′ 6 is straightforward.…”

“…As a test when all substrates are saturating, k′ 6 can be calculated from the relationship of k cat and the net rate constants of the catalytic sequence according to eq 13.…”

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

“…The kinetic competence of the analogous HE-TPP radical in the reaction of M. thermoacetica PFOR was subsequently demonstrated (10). Thus, in the absence of CoA, the radical appears at a rate ∼ 3 fold greater than k cat , and upon addition of CoA, the radical decays at a rate ≫ k cat giving rise to the normal products of the reaction (10). Despite differences in subunit composition and in the number of [4Fe-4S] clusters among PFOR's from different organisms, it is likely that oxidation of the HE-TPP intermediate occurs in two single-electron steps in most, if not all PFOR's (10).…”

“…Thus, in the absence of CoA, the radical appears at a rate ∼ 3 fold greater than k cat , and upon addition of CoA, the radical decays at a rate ≫ k cat giving rise to the normal products of the reaction (10). Despite differences in subunit composition and in the number of [4Fe-4S] clusters among PFOR's from different organisms, it is likely that oxidation of the HE-TPP intermediate occurs in two single-electron steps in most, if not all PFOR's (10). This view is supported by careful inspection of EPR spectra published for the HE-TPP radical intermediate of PFOR's from M. thermoaceticum (10), D. africanus (11), H. halobium (12), and Methanosarcina bakeri (13).…”

Pulsed Electron Paramagnetic Resonance Experiments Identify the Paramagnetic Intermediates in the Pyruvate Ferredoxin Oxidoreductase Catalytic Cycle

“…4,8 A subsequent electron transfer from the HE-TPP radical leaves two reduced [4Fe-4S] 1+ clusters. The rate of this redox reaction and, hence, the stability of the HE-TPP radical depends strongly on the presence of CoA.…”