Advertisement

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

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

Abstract

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.

Keywords

Nickel Iron–sulfur clusters Metabolic channeling 

Notes

Acknowledgement

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

References

  1. 1.
    Lindahl PA (2002) Biochemistry 41:2097–2105CrossRefPubMedGoogle Scholar
  2. 2.
    Riordan CG (2004) J Biol Inorg Chem 9:509–510Google Scholar
  3. 3.
    Riordan CG (2004) J Biol Inorg Chem 9:542–549PubMedGoogle Scholar
  4. 4.
    Lindahl PA (2004) J Biol Inorg Chem 9:516–524CrossRefPubMedGoogle Scholar
  5. 5.
    Brunold TC. (2004) J Biol Inorg Chem 9:533–541CrossRefPubMedGoogle Scholar
  6. 6.
    Volbeda A, Fontecilla-Camps JC (2004) J Biol Inorg Chem 9:525–532CrossRefPubMedGoogle Scholar
  7. 7.
    Drennan CL, Doukov TI, Ragsdale SW (2004) J Biol Inorg Chem 9:511–515PubMedCrossRefGoogle Scholar
  8. 8.
    Doukov TI, Iverson TM, Seravalli J, Ragsdale SW, Drennan CL (2002) Science 298:567–572PubMedCrossRefGoogle Scholar
  9. 9.
    Darnault C, Volbeda A, Kim EJ, Legrand P, Vernede X, Lindahl PA, Fontecilla-Camps JC (2003) Nat Struct Biol 10:271–279CrossRefPubMedGoogle Scholar
  10. 10.
    Ragsdale SW, Clark JE, Ljungdahl LG, Lundie LL, Drake HL (1983) J Biol Chem 258:2364–2369PubMedGoogle Scholar
  11. 11.
    Dobbek H, Svetlitchnyi V, Gremer L, Huber R, Meyer O (2001) Science 293:1281–1285CrossRefPubMedGoogle Scholar
  12. 12.
    Drennan CL, Heo JY, Sintchak MD, Schreiter E, Ludden PW (2001) Proc Natl Acad Sci USA 98:11973–11978CrossRefPubMedGoogle Scholar
  13. 13.
    Kim EJ, Feng J, Bramlet MR, Lindahl PA (2004) Biochemistry 43:5725–5734Google Scholar
  14. 14.
    Loke HK, Tan X, Lindahl PA (2002) J Am Chem Soc 124:8667–8672CrossRefPubMedGoogle Scholar
  15. 15.
    Svetlitchnyi V, Dobbek H, Meyer-Klaucke W, Meins T, Thiele B, Romer P, Huber R, Meyer O (2004) Proc Natl Acad Sci USA 101:446–451CrossRefPubMedGoogle Scholar
  16. 16.
    Gencic S, Grahame DA (2003) J Biol Chem 278:6101–6110CrossRefPubMedGoogle Scholar
  17. 17.
    Maynard EL, Lindahl PA (1999) J Am Chem Soc121:9221–9222CrossRefGoogle Scholar
  18. 18.
    Seravalli J, Ragsdale SW (2000) Biochemistry 39:1274–1277CrossRefPubMedGoogle Scholar
  19. 19.
    Raushel FM, Thoden JB, Holden HM (2003) Acc Chem Res 36:539–548CrossRefPubMedGoogle Scholar
  20. 20.
    Tan X, Bramlett MR, Lindahl PA (2004) J Am Chem Soc 126:5954–5955CrossRefPubMedGoogle Scholar
  21. 21.
    Maynard EL, Sewell C, Lindahl PA (2001) J Am Chem Soc 123:4697–4703CrossRefPubMedGoogle Scholar
  22. 22.
    Tan X, Loke HK, Fitch S, Lindahl PA (2005) J Am Chem Soc 127:5833–5839CrossRefPubMedGoogle Scholar
  23. 23.
    Volbeda A, Fontecilla-Camps JC (2005) Dalton Trans 21:3443–3450CrossRefPubMedGoogle Scholar
  24. 24.
    Lundie LL, Drake HL (1984) J Bacteriol 159:700–703PubMedGoogle Scholar
  25. 25.
    Loke HK, Bennett GN, Lindahl PA (2000) Proc Natl Acad Sci USA 97:12530–12535CrossRefPubMedGoogle Scholar
  26. 26.
    Pelley JW, Garner CW, Little GH (1978) Anal Biochem 86:341–343CrossRefPubMedGoogle Scholar
  27. 27.
    Shin W, Lindahl PA (1992) Biochim Biophys Acta 1161:317–322Google Scholar
  28. 28.
    Tan X, Sewell C, Lindahl PA (2002) J Am Chem Soc 124:6277–6284CrossRefPubMedGoogle Scholar
  29. 29.
    Xia JQ, Lindahl PA (1995) Biochemistry 34:6037–6042CrossRefPubMedGoogle Scholar
  30. 30.
    Lindahl PA, Münck E, Ragsdale SW (1990) J Biol Chem 265:3873–3879PubMedGoogle Scholar
  31. 31.
    Craft JL, Ludden PW, Brunold TC (2002) Biochemistry 41:1681–1688CrossRefPubMedGoogle Scholar
  32. 32.
    Ragsdale SW, Wood HG (1985) J Biol Chem 260:3970–3977PubMedGoogle Scholar
  33. 33.
    Collaborative Computational Project, Number 4 (1994) Acta Crystallogr D 50:760–763Google Scholar
  34. 34.
    Lawrence MC, Bourke P (2000) J Appl Crystallogr 33:990–991CrossRefGoogle Scholar
  35. 35.
    Kraulis PJ (1991) J Appl Crystallogr 24:946–950CrossRefGoogle Scholar
  36. 36.
    Merrit EA, Bacon DJ (1997) Methods Enzymol 277:520–524Google Scholar

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

Personalised recommendations