Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle

Andrei V. Astashkin, Javier Seravalli, Steven O. Mansoorabadi, George H. Reed, Stephen W. Ragsdale

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.

Original languageEnglish (US)
Pages (from-to)3888-3889
Number of pages2
JournalJournal of the American Chemical Society
Volume128
Issue number12
DOIs
StatePublished - Mar 29 2006

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Pyruvate Synthase
Thiamine Pyrophosphate
Electron Spin Resonance Spectroscopy
Paramagnetic resonance
Electrons
Pyruvic Acid
Experiments
Anaerobiosis
Ferredoxins
Decarboxylation
Eukaryota
Metabolism
Bacteria
Crystal structure
Oxidation
Oxidoreductases

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

Cite this

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle. / Astashkin, Andrei V.; Seravalli, Javier; Mansoorabadi, Steven O.; Reed, George H.; Ragsdale, Stephen W.

In: Journal of the American Chemical Society, Vol. 128, No. 12, 29.03.2006, p. 3888-3889.

Research output: Contribution to journalArticle

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