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Mechanism of pyruvate ferredoxin oxidoreductase and role of the (4Fe-4S) cluster of the corrinoid iron sulfur protein in acetyl COA synthesis by the Wood Ljungdahl pathway

Saurabh Prabhakar Menon, University of Nebraska - Lincoln

Abstract

Pyruvate ferredoxin oxidoreductase (PFOR) catalyzes the oxidative decarboxylation of pyruvate to acetyl CoA and CO$\sb2.$ By using rapid freeze quench EPR and stopped-flow spectroscopy the three elementary steps that constitute the first reductive half reaction of the PFOR mechanism were elucidated. A hydroxyethyl-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. The CO$\sb2$ produced in the PFOR reaction was shown to be reduced to CO by CODH/ACS; subsequently, the CO generated binds in the ACS active site at a unique metal center to form a paramagnetic adduct the nickel iron carbon species. This bound carbonyl group then combines with a bound methyl group and coenzyme A to generate a third molecule of acetyl CoA. Two common intermediates were shown to be formed in acetyl CoA synthesis from CO$\sb2$: CO and the paramagnetic nickel iron carbon species making the Wood Ljungdahl pathway the first pathway in which CO is produced and then used as a metabolic intermediate. Both PFOR and CODH/ACS were also shown to be able to reduce protons to H$\sb2$ and at much lower rates, oxidize H$\sb2$ to protons and electrons. PFOR and CODH/ACS when incubated with their substrates/electron donors displayed self oxidation in the absence of an electron acceptor. The observed level of hydrogenase activity is physiologically relevant because it accounts for the rate of hydrogen production by growing cultures of Clostridium thermoaceticum. Another protein the corrinoid iron sulfur protein (CFeSP) which contains a cobalt ion housed in a corrinoid cofactor and an iron sulfur cluster catalyzes two key steps in the Wood Ljungdahl pathway. We have elucidated the role of the (4Fe-4S) cluster by site directed mutagenesis, and have demonstrated that it is required only in the reductive activation cycle that couples the oxidation of substrates like CO to the reduction of Co$\sp{+2}.$

Subject Area

Biochemistry

Recommended Citation

Menon, Saurabh Prabhakar, "Mechanism of pyruvate ferredoxin oxidoreductase and role of the (4Fe-4S) cluster of the corrinoid iron sulfur protein in acetyl COA synthesis by the Wood Ljungdahl pathway" (1997). ETD collection for University of Nebraska-Lincoln. AAI9815900.
https://digitalcommons.unl.edu/dissertations/AAI9815900

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