Kinetic and crystallographic studies of a redesigned manganese-binding site in cytochrome c peroxidase

  • Thomas D. Pfister
  • Amir Y. Mirarefi
  • Alan J. Gengenbach
  • Xuan Zhao
  • Connor Danstrom
  • Nicole Conatser
  • Yi-Gui Gao
  • Howard Robinson
  • Charles F. Zukoski
  • Andrew H.-J. Wang
  • Yi Lu
Original Paper

Abstract

Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a MnII-binding site was designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215–222, 1997; Gengenbach et al. in Biochemistry 38:11425–11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k cat/K M) than the variant in the original design, mostly due to a stronger K M of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with CoII at the designed MnII site were also obtained. The metal ion in the engineered metal-binding site overlays well with MnII bound in MnP, suggesting that this variant is the closest structural model of the MnII-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal–ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (CoII rather than MnII) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.

Keywords

Manganese oxidation Metal-binding site Protein engineering Metalloprotein Biomimetic 

Abbreviations

CcP

Cytochrome c peroxidase from Saccharomyces cerevisiae

CcP(MI)

Recombinant yeast CcP containing Met-Ile at the N-terminus in addition to the normal wild-type yeast CcP sequence

EPR

Electron paramagnetic resonance

Compound I

Ferryl (FeIV-oxo) heme and porphyrin cation radical

Compound I•

Ferryl (FeIV-oxo) heme and Trp radical

Compound II

Ferryl (FeIV-oxo) heme

MnCcP

CcP(G41E, V45E, H181D)

MnCcP2

CcP(G41E, V45E, W51F, H181D, W191F)

MnCcP.1

CcP(D37E, V45E, H181E)

MnP

Manganese peroxidase

MP6.8

CcP(D37E, P44D, V45D)

PC

Phanerochaete chrysosporium

WTCcP

Wild-type yeast CcP

Notes

Acknowledgements

This material is based upon work supported by the National Institute of General Medical Sciences in the National Institute of Health (GM62211 to Y.L.).

Supplementary material

References

  1. 1.
    Lippard SJ, Berg JM (1994) Principles of bioinorganic chemistry. University Science Books, Mill ValleyGoogle Scholar
  2. 2.
    Bertini I, Gray HB, Lippard SJ, Valentine JS (1994) Bioinorganic chemistry. University Science Books, SausalitoGoogle Scholar
  3. 3.
    Holm RH, Kennepohl P, Solomon EI (1996) Chem Rev 96:2239–2314PubMedCrossRefGoogle Scholar
  4. 4.
    Thomson AJ, Gray HB (1998) Curr Opin Chem Biol 2:155–158PubMedCrossRefGoogle Scholar
  5. 5.
    DeGrado WF, Summa CM, Pavone V, Nastri F, Lombardi A (1999) Annu Rev Biochem 68:779–819PubMedCrossRefGoogle Scholar
  6. 6.
    Lu Y, Berry SM, Pfister TD (2001) Chem Rev 101:3047–3080PubMedCrossRefGoogle Scholar
  7. 7.
    Watanabe Y (2002) Curr Opin Chem Biol 6:208–216PubMedCrossRefGoogle Scholar
  8. 8.
    Reedy CJ, Gibney BR (2004) Chem Rev 104:617–649PubMedCrossRefGoogle Scholar
  9. 9.
    Sundaramoorthy M, Kishi K, Gold MH, Poulos TL (1994) J Biol Chem 269:32759–32767PubMedGoogle Scholar
  10. 10.
    Sundaramoorthy M, Youngs HL, Gold MH, Poulos TL (2005) Biochemistry 44:6463–6470PubMedCrossRefGoogle Scholar
  11. 11.
    Gold MH, Wariishi H, Valli K (1989) ACS Symp Ser 389:127–140CrossRefGoogle Scholar
  12. 12.
    Tien M, Cai D (1990) Biol Oxid Syst Proc Symp 1:433–451Google Scholar
  13. 13.
    Gold MH, Youngs HL, Gelpke MD S (2000) Met Ions Biol Syst 37:559–586Google Scholar
  14. 14.
    Dunford HB (1999) Heme peroxidases. Wiley-VCH, New YorkGoogle Scholar
  15. 15.
    Kishi K, Kusters-van Someren M, Mayfield MB, Sun J, Loehr TM, Gold MH (1996) Biochemistry 35:8986–8994PubMedCrossRefGoogle Scholar
  16. 16.
    Sundaramoorthy M, Kishi K, Gold MH, Poulos TL (1997) J Biol Chem 272:17574–17580PubMedCrossRefGoogle Scholar
  17. 17.
    Kusters-van Someren M., Kishi K, Lundell T, Gold MH (1995) Biochemistry 34:10620–10627PubMedCrossRefGoogle Scholar
  18. 18.
    Gelpke MD, Youngs HL, Gold MH (2000) Eur J Biochem 267:7038–7045PubMedCrossRefGoogle Scholar
  19. 19.
    Youngs HL, Sollewijn Gelpke MD, Li D, Sundaramoorthy M, Gold MH (2001) Biochemistry 40:2243–2250PubMedCrossRefGoogle Scholar
  20. 20.
    Yeung BK, Wang X, Sigman JA, Petillo PA, Lu Y (1997) Chem Biol 4:215–221PubMedCrossRefGoogle Scholar
  21. 21.
    Wilcox SK, Putnam CD, Sastry M, Blankenship J, Chazin WJ, McRee DE, Goodin DB (1998) Biochemistry 37:16853–16862PubMedCrossRefGoogle Scholar
  22. 22.
    Wang X, Lu Y (1999) Biochemistry 38:9146–9157PubMedCrossRefGoogle Scholar
  23. 23.
    Gengenbach A, Syn S, Wang X, Lu Y (1999) Biochemistry 38:11425–11432PubMedCrossRefGoogle Scholar
  24. 24.
    Gengenbach A, Wang X, Lu Y (2001) In: Argyropoulos DS (ed) Oxidative delignification chemistry, fundamentals and catalysis, ACS symp. ser. vol 785. American Chemical Society, Washington, pp 487–500Google Scholar
  25. 25.
    Mester T, Tien M (2001) Biochem Biophys Res Commun 284:723–728PubMedCrossRefGoogle Scholar
  26. 26.
    Hunter CL, Maurus R, Mauk MR, Lee H, Raven EL, Tong H, Nguyen N, Smith M, Brayer GD, Mauk AG (2003) Proc Natl Acad Sci USA 100:3647–3652PubMedCrossRefGoogle Scholar
  27. 27.
    Poulos TL, Fenna RE (1994) In: Sigel H, Sigel A (eds) Metal ions in biological systems, vol 30. Dekker, New York, pp 25–75Google Scholar
  28. 28.
    English AM, Tsaprailis G (1995) Adv Inorg Chem 43:79–125Google Scholar
  29. 29.
    Erman JE, Vitello LB (1998) J Biochem Mol Biol 31:307–327Google Scholar
  30. 30.
    Poulos TL, Kraut J (1980) J Biol Chem 255:8199–8205PubMedGoogle Scholar
  31. 31.
    Goodin DB, Mauk AG, Smith M (1986) Proc Natl Acad Sci USA 83:1295–1299PubMedCrossRefGoogle Scholar
  32. 32.
    Sivaraja M, Goodin DB, Smith M, Hoffman BM (1989) Science 245:738–740PubMedCrossRefGoogle Scholar
  33. 33.
    Ivancich A, Dorlet P, Goodin DB, Un S (2001) J Am Chem Soc 123:5050–5058PubMedCrossRefGoogle Scholar
  34. 34.
    Nelson DP, Kiesow LA (1972) Anal Biochem 49:474–478PubMedCrossRefGoogle Scholar
  35. 35.
    Pfister TD, Gengenbach AJ, Syn S, Lu Y (2001) Biochemistry 40:14942–14951PubMedCrossRefGoogle Scholar
  36. 36.
    De Duve C (1948) Acta Chem Scand 2:264–289CrossRefGoogle Scholar
  37. 37.
    Morrison M, Horie S (1965) Anal Biochem 12:77–82PubMedCrossRefGoogle Scholar
  38. 38.
    Wariishi H, Valli K, Gold MH (1992) J Biol Chem 267:23688–23695PubMedGoogle Scholar
  39. 39.
    Kuan IC, Tien M (1993) Proc Natl Acad Sci USA 90:1242–1246PubMedCrossRefGoogle Scholar
  40. 40.
    Khindaria A, Barr D, Aust SD (1995) Biochemistry 34:7773–7779PubMedCrossRefGoogle Scholar
  41. 41.
    Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326Google Scholar
  42. 42.
    Navaza J (1994) Acta Crystallogr Sect A 50:157–163CrossRefGoogle Scholar
  43. 43.
    Sheldrick GM, Schneider TR (1997) Methods Enzymol 277:319–343PubMedGoogle Scholar
  44. 44.
    Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Acta Crystallogr Sect A 47:110–119CrossRefGoogle Scholar
  45. 45.
    Brunger AT (1992) XPLOR. Yale University, New HavenGoogle Scholar
  46. 46.
    Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Crystallogr 26:283–291CrossRefGoogle Scholar
  47. 47.
    Finzel BC, Poulos TL, Kraut J (1984) J Biol Chem 259:13027–13036PubMedGoogle Scholar
  48. 48.
    Fishel LA, Farnum MF, Mauro JM, Miller MA, Kraut J, Liu Y, Tan XL, Scholes CP (1991) Biochemistry 30:1986–1996PubMedCrossRefGoogle Scholar
  49. 49.
    Harris RZ, Wariishi H, Gold MH, Ortiz de Montellano PR (1991) J Biol Chem 266:8751–8758PubMedGoogle Scholar
  50. 50.
    Youngs HL, Sundaramoorthy M, Gold MH (2000) Eur J Biochem 267:1761–1769PubMedCrossRefGoogle Scholar
  51. 51.
    Loo S, Erman JE (1975) Biochemistry 14:3467–3470PubMedCrossRefGoogle Scholar
  52. 52.
    Vitello LB, Huang M, Erman JE (1990) Biochemistry 29:4283–4288PubMedCrossRefGoogle Scholar
  53. 53.
    Erman JE, Vitello LB, Miller MA, Shaw A, Brown KA, Kraut J (1993) Biochemistry 32:9798–9806PubMedCrossRefGoogle Scholar
  54. 54.
    Glenn JK, Gold MH (1985) Arch Biochem Biophys 242:329–341PubMedCrossRefGoogle Scholar
  55. 55.
    Barrows TP, Bhaskar B, Poulos TL (2004) Biochemistry 43:8826–8834PubMedCrossRefGoogle Scholar
  56. 56.
    Shannon RD (1976) Acta Crystallogr Sect A 32:751–767CrossRefGoogle Scholar
  57. 57.
    See RF, Kruse RA, Strub WM (1998) Inorg Chem 37:5369–5375CrossRefGoogle Scholar
  58. 58.
    Eshaghi S, Niegowski D, Kohl A, Molina DM, Lesley SA, Nordlund P (2006) Science 313:354–357PubMedCrossRefGoogle Scholar
  59. 59.
    Gelpke MDS, Moeenne-Loccoz P, Gold MH (1999) Biochemistry 38:11482–11489CrossRefGoogle Scholar
  60. 60.
    Camarero S, Sarkar S, Ruiz-Duenas FJ, Martinez MJ, Martinez AT (1999) J Biol Chem 274:10324–10330PubMedCrossRefGoogle Scholar
  61. 61.
    Mayfield MB, Kishi K, Alic M, Gold MH (1994) Appl Environ Microbiol 60:4303–4309PubMedGoogle Scholar

Copyright information

© SBIC 2006

Authors and Affiliations

  • Thomas D. Pfister
    • 1
  • Amir Y. Mirarefi
    • 2
  • Alan J. Gengenbach
    • 3
  • Xuan Zhao
    • 3
  • Connor Danstrom
    • 3
  • Nicole Conatser
    • 3
  • Yi-Gui Gao
    • 4
  • Howard Robinson
    • 5
  • Charles F. Zukoski
    • 2
  • Andrew H.-J. Wang
    • 6
  • Yi Lu
    • 1
    • 3
  1. 1.Department of BiochemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  2. 2.Department of Chemical and Biomolecular EngineeringUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  3. 3.Department of ChemistryUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  4. 4.School of Chemical Sciences Biocrystallization ServiceUniversity of Illinois at Urbana-ChampaignUrbanaUSA
  5. 5.Department of BiologyBrookhaven National LaboratoryUptonUSA
  6. 6.Institute of Biological Chemistry and Core Facility for Protein CrystallographyAcademia SinicaTaipeiTaiwan

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