EPR and infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO

Simon J. George, Javier Seravalli, Stephen W. Ragsdale

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.

Original languageEnglish (US)
Pages (from-to)13500-13501
Number of pages2
JournalJournal of the American Chemical Society
Volume127
Issue number39
DOIs
StatePublished - Oct 5 2005

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Coenzymes
Acetyl Coenzyme A
Carbon Monoxide
Paramagnetic resonance
Enzymes
Infrared radiation
Atoms
carbon monoxide dehydrogenase
Carbon dioxide
Sulfur
Carbon Dioxide
Cysteine
Catalytic Domain
Carbon monoxide
Escherichia coli
Infrared spectroscopy
Archaea
Bacteria
Wood
Tunnels

ASJC Scopus subject areas

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

Cite this

EPR and infrared spectroscopic evidence that a kinetically competent paramagnetic intermediate is formed when acetyl-coenzyme A synthase reacts with CO. / George, Simon J.; Seravalli, Javier; Ragsdale, Stephen W.

In: Journal of the American Chemical Society, Vol. 127, No. 39, 05.10.2005, p. 13500-13501.

Research output: Contribution to journalArticle

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AB - Carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) is a bifunctional enzyme which enables archaea and bacteria to grow autotrophically on CO and hydrogen/carbon dioxide using the Wood-Ljundahl pathway. CO produced from reduction of carbon dioxide by CODH is transferred to the active site of ACS through an intramolecular tunnel, where it combines with Coenzyme A and a methyl cation to produce acetyl-CoA. The active site of ACS contains a single [4Fe-4S] cluster bridged by a cysteine sulfur atom to a binuclear center. The binuclear center is composed of two Ni atoms bridged by two separate cysteine sulfurs. The Ni site attached to the [4Fe-4S] is referred to as proximal Ni, while the other Ni atom, which assumes a square-planar geometry, is referred to as the distal site. We report the characterization of the carbonylated form of highly active (0.67 spins/mol) heterologously expressed monomeric ACS from C. hydrogenoformans in E. coli by rapid-freeze quench EPR (RFQ-EPR) and stopped-flow infrared (SF-IR) spectroscopies. The reaction of ACS with CO produces a single metal-carbonyl species whose formation rate, measured by SF-IR, correlates with the rate of formation, measured by RFQ-EPR, of the paramagnetic state of the enzyme (NiFeC species). These results indicate that the NiFeC species is the predominant form observed in solution when ACS reacts with CO. The NiFeC species contains the proximal Ni in the +1 redox state and the [4Fe-4S] cluster in the 2+ state, thus there is no evidence for either a Ni(0) or a Ni(II) state in the active carbonylated form of the enzyme.

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