Mechanism of the Clostridium thermoaceticum pyruvate:ferredoxin oxidoreductase: Evidence for the common catalytic intermediacy of the hydroxyethylthiamine pyropyrosphate radical

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 Clostridium thermoaceticum 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 similar to 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 die 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 similar to 4400-fold.