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 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.
Menon
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Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Ragsdale
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D003013:Clostridium / Q000201:enzymology*; D003065:Coenzyme A / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000378:metabolism; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D007658:Ketone Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010269:Paraquat / Q000378:metabolism; D011485:Protein Binding; D050743:Pyruvate Synthase; D019289:Pyruvic Acid / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives* / Q000378:metabolism
D005609:Free Radicals; D007506:Iron-Sulfur Proteins; C047467:2-(1-hydroxyethyl)thiamine pyrophosphate; D019289:Pyruvic Acid; D007501:Iron; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; D010269:Paraquat; D013835:Thiamine Pyrophosphate; D003065:Coenzyme A