TY - JOUR
T1 - Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle
AU - Astashkin, Andrei V.
AU - Seravalli, Javier
AU - Mansoorabadi, Steven O.
AU - Reed, George H.
AU - Ragsdale, Stephen W.
N1 - Copyright:
Copyright 2010 Elsevier B.V., All rights reserved.
PY - 2006/3/29
Y1 - 2006/3/29
N2 - 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.
AB - 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.
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U2 - 10.1021/ja0585275
DO - 10.1021/ja0585275
M3 - Article
C2 - 16551078
AN - SCOPUS:33645460049
SN - 0002-7863
VL - 128
SP - 3888
EP - 3889
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 12
ER -