Enzymatic and electron paramagnetic resonance studies of anabolic pyruvate synthesis by pyruvate: ferredoxin oxidoreductase from Hydrogenobacter thermophilus.
Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thiamine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO(2). The thermophilic, obligate chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO(2) via the reductive tricarboxylic acid cycle. In this cycle, POR acts as pyruvate synthase catalyzing the reverse reaction (i.e. reductive carboxylation of acetyl-CoA) to form pyruvate. The pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant. Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin. Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood. In the present study, we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxoglutarate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding pyruvate synthesis by POR. We demonstrate that H. thermophilus POR catalyzes pyruvate synthesis from acetyl-CoA and CO(2), confirming the operation of the reductive tricarboxylic acid cycle in this bacterium. We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate radical in both reactions. The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed.
Ikeda
Takeshi
T
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Yamamoto
Masahiro
M
Arai
Hiroyuki
H
Ohmori
Daijiro
D
Ishii
Masaharu
M
Igarashi
Yasuo
Y
D000105:Acetyl Coenzyme A / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002245:Carbon Dioxide / Q000378:metabolism; D002952:Citric Acid Cycle; D004578:Electron Spin Resonance Spectroscopy; D004734:Energy Metabolism; D007658:Ketone Oxidoreductases / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D050743:Pyruvate Synthase / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D002245:Carbon Dioxide; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D007770:L-Lactate Dehydrogenase; D007658:Ketone Oxidoreductases; D050743:Pyruvate Synthase; C023096:2-oxoglutarate synthase