Function of the tunnel in acetylcoenzyme A synthase/carbon monoxide dehydrogenase

  • Xiangshi Tan
  • Anne Volbeda
  • Juan C. Fontecilla-Camps
  • Paul A. Lindahl
Original Paper


Acetylcoenzyme A synthase/carbon monoxide dehydrogenase (ACS/CODH) contains two Ni–Fe–S active-site clusters (called A and C) connected by a tunnel through which CO and CO2 migrate. Site-directed mutants A578C, L215F, and A219F were designed to block the tunnel at different points along the region between the two C-clusters. Two other mutant proteins F70W and N101Q were designed to block the region that connects the tunnel at the ββ interface with a water channel also located at that interface. Purified mutant proteins were assayed for Ni/Fe content and examined by electron paramagnetic resonance spectroscopy. Analyses indicate that same metal clusters found in wild-type (WT) ACS/CODH (i.e., the A-, B-, C-, and probably D-clusters) are properly assembled in the mutant enzymes. Stopped-flow kinetics revealed that these centers in the mutants are rapidly reducible by dithionite but are only slowly reducible by CO, suggesting an impaired ability of CO to migrate through the tunnel to the C-cluster. Relative to the WT enzyme, mutant proteins exhibited little CODH or ACS activity (using CO2 as a substrate). Some ACS activity was observed when CO was a substrate, but not the cooperative CO inhibition effect characteristic of WT ACS/CODH. These results suggest that CO and CO2 enter and exit the enzyme at the water channel along the ββ subunit interface. They also suggest two pathways for CO during synthesis of acetylcoenzyme A, including one in which CO enters the enzyme and migrates through the tunnel before binding at the A-cluster, and another in which CO binds the A-cluster directly from the solvent.


Nickel Iron–sulfur clusters Metabolic channeling 



This work was supported by the National Institute of Health (GM46441).


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Copyright information

© SBIC 2006

Authors and Affiliations

  • Xiangshi Tan
    • 1
  • Anne Volbeda
    • 2
  • Juan C. Fontecilla-Camps
    • 2
  • Paul A. Lindahl
    • 1
  1. 1.Departments of Chemistry and Biochemistry & BiophysicsTexas A&M UniversityCollege StationUSA
  2. 2.Laboratoire de Cristallographie et Cristallogenèse des ProtéinesInstitut de Biologie Structurale ‘Jean-Pierre Ebel’, CEA, UJF, CNRSGrenoble Cedex 1France

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