Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum: Kinetic characterization of the intermediates

Javier Seravalli, Manoj Kumar, Wei Ping Lu, Stephen W. Ragsdale

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Abstract

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV- visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., and Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., and Lindahl, P. A [1996) Biochemistry 35, 8371-8380] that the C(red2) form of cluster C is two electrons more reduced than the C(red1) form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [C(red1),B(ox),X(ox)] state to yield a C(red1)-CO complex; (ii) two-electron reduction of C(red1) to C(red2) concerted with CO2 release; (iii) binding of a second CO molecule to the [C(red2),B(ox),X(ox)] state to form a C(red2)-CO complex; (iv) electron transfer from C(red2)-CO to cluster B to form [C(red2),B(red),X(red)] with concerted release of the second CO2. Step iii competes with internal electron transfer from C(red2) to B(ox) and X(ox). At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. X(ox) is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The C(red2) state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.

Original languageEnglish (US)
Pages (from-to)11241-11251
Number of pages11
JournalBiochemistry
Volume36
Issue number37
DOIs
StatePublished - Sep 16 1997

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carbon monoxide dehydrogenase
Clostridium
Acetyl Coenzyme A
Carbon Monoxide
Electrons
Oxidation
Kinetics
Electron Spin Resonance Spectroscopy
Titration
Paramagnetic resonance
Ferredoxins
Biochemistry
Spectrum Analysis
Corrinoids
Metals
Spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum : Kinetic characterization of the intermediates. / Seravalli, Javier; Kumar, Manoj; Lu, Wei Ping; Ragsdale, Stephen W.

In: Biochemistry, Vol. 36, No. 37, 16.09.1997, p. 11241-11251.

Research output: Contribution to journalArticle

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abstract = "Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV- visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., and Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., and Lindahl, P. A [1996) Biochemistry 35, 8371-8380] that the C(red2) form of cluster C is two electrons more reduced than the C(red1) form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [C(red1),B(ox),X(ox)] state to yield a C(red1)-CO complex; (ii) two-electron reduction of C(red1) to C(red2) concerted with CO2 release; (iii) binding of a second CO molecule to the [C(red2),B(ox),X(ox)] state to form a C(red2)-CO complex; (iv) electron transfer from C(red2)-CO to cluster B to form [C(red2),B(red),X(red)] with concerted release of the second CO2. Step iii competes with internal electron transfer from C(red2) to B(ox) and X(ox). At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. X(ox) is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The C(red2) state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.",
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T1 - Mechanism of carbon monoxide oxidation by the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum

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AU - Seravalli, Javier

AU - Kumar, Manoj

AU - Lu, Wei Ping

AU - Ragsdale, Stephen W.

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N2 - Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV- visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., and Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., and Lindahl, P. A [1996) Biochemistry 35, 8371-8380] that the C(red2) form of cluster C is two electrons more reduced than the C(red1) form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [C(red1),B(ox),X(ox)] state to yield a C(red1)-CO complex; (ii) two-electron reduction of C(red1) to C(red2) concerted with CO2 release; (iii) binding of a second CO molecule to the [C(red2),B(ox),X(ox)] state to form a C(red2)-CO complex; (iv) electron transfer from C(red2)-CO to cluster B to form [C(red2),B(red),X(red)] with concerted release of the second CO2. Step iii competes with internal electron transfer from C(red2) to B(ox) and X(ox). At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. X(ox) is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The C(red2) state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.

AB - Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) from Clostridium thermoaceticum catalyzes (i) the synthesis of acetyl-CoA from a methylated corrinoid protein, CO, and coenzyme A and (ii) the oxidation of CO to CO2. CO oxidation occurs at a Ni- and FeS-containing center known as cluster C. Electrons are transferred from cluster C to a separate metal center, cluster B, to external acceptors like ferredoxin. In the work described here, we performed reductive titrations of CODH/ACS with CO and sodium dithionite and monitored the reaction by electron paramagnetic resonance (EPR) spectroscopy. We also performed pre-steady-state kinetic studies by rapid freeze-quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics. Redox titrations of CODH/ACS revealed the existence of a UV- visible and EPR-silent electron acceptor denoted center S that does not appear to be associated with any of the other metal centers in the protein. Our results support the previous proposals [Anderson, M. E., and Lindahl, P. A. (1994) Biochemistry 33, 8702-8711; Anderson, M. E., and Lindahl, P. A [1996) Biochemistry 35, 8371-8380] that the C(red2) form of cluster C is two electrons more reduced than the C(red1) form. The combined results from titrations and pre-steady-state studies were used to formulate a mechanism for CO oxidation, composed of the following steps: (i) CO binding to the [C(red1),B(ox),X(ox)] state to yield a C(red1)-CO complex; (ii) two-electron reduction of C(red1) to C(red2) concerted with CO2 release; (iii) binding of a second CO molecule to the [C(red2),B(ox),X(ox)] state to form a C(red2)-CO complex; (iv) electron transfer from C(red2)-CO to cluster B to form [C(red2),B(red),X(red)] with concerted release of the second CO2. Step iii competes with internal electron transfer from C(red2) to B(ox) and X(ox). At high CO concentrations, step iii is favored, whereas at low concentrations, only one CO molecule per turnover binds and undergoes oxidation. Closure of the catalytic cycle involves electron transfer from reduced enzyme to an electron acceptor protein, like ferredoxin. X(ox) is a yet-uncharacterized electron acceptor that may be an intermediate in the reduction of center S. The C(red2) state appears to be the predominant state of cluster C during steady-state turnover. The rate-determining step for the first half-reaction is step iv, while during steady-state turnover, it appears to be electron transfer to external electron acceptors.

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