Advertisement

Protein recognition in ferredoxin–P450 electron transfer in the class I CYP199A2 system from Rhodopseudomonas palustris

  • Stephen G. BellEmail author
  • Feng Xu
  • Eachan O. D. Johnson
  • Ian M. Forward
  • Mark Bartlam
  • Zihe RaoEmail author
  • Luet-Lok Wong
Original Paper

Abstract

CYP199A2 from Rhodopseudomonas palustris CGA009 is a heme monooxygenase that catalyzes the oxidation of para-substituted benzoic acids. CYP199A2 activity is reconstituted by a class I electron transfer chain consisting of the associated [2Fe–2S] ferredoxin palustrisredoxin (Pux) and a flavoprotein palustrisredoxin reductase (PuR). Another [2Fe–2S] ferredoxin, palustrisredoxin B (PuxB; RPA3956) has been identified in the genome. PuxB shares sequence identity and motifs with vertebrate-type ferredoxins involved in Fe–S cluster assembly but also 50% identity with Pux and it mediates electron transfer from PuR to CYP199A2, albeit with lower steady-state turnover activity: 99 nmol (nmol P450)−1min−1 for 4-methoxybenzoic acid oxidation compared with 1,438 nmol (nmol P450)−1 min−1 for Pux. This difference mainly arises from weak CYP199A2–PuxB binding (K m 34.3 vs. 0.45 μM for Pux) rather than slow electron transfer (k cat 19.1 vs. 37.9 s−1 for Pux). Comparison of the 2.0-Å-resolution crystal structure of the PuxB A105R mutant with other vertebrate-type, P450-associated ferredoxins revealed similar protein folds but also significant differences in some loop regions. Therefore, PuxB offers a platform for studying ferredoxin–P450 recognition in class I P450 systems. Substitution of PuxB residues at key locations with those in Pux shows that Ala42, Cys43, and Ala44 in the [2Fe–2S] cluster binding loop and Met66 are important in electron transfer from PuxB to CYP199A2, whereas Phe73 and the C-terminal Ala105 were involved in both protein binding and electron transfer.

Keywords

Cytochrome P450 Ferredoxin Electron transfer Rhodopseudomonas palustris Mutagenesis 

Notes

Acknowledgements

The authors would like to thank the reviewers for helpful suggestions that helped improve the manuscript. This work was supported by the Higher Education Funding Council for England, the Engineering and Physical Sciences Research Council, and the Biotechnology and Biological Sciences Research Council (EP-D048559-1), UK, and the National Science Foundation of China (30221003).

Supplementary material

775_2009_604_MOESM1_ESM.pdf (3.3 mb)
Supplementary material 1 (PDF 3372 kb)

References

  1. 1.
    Ortiz de Montellano PR (ed) (2005) Cytochrome P450: structure, mechanism, and biochemistry. Kluwer/Plenum Press, New YorkGoogle Scholar
  2. 2.
    Guengerich FP (2001) Chem Res Toxicol 14:611–650. doi: 10.1021/tx0002583 CrossRefPubMedGoogle Scholar
  3. 3.
    Denisov IG, Makris TM, Sligar SG, Schlichting I (2005) Chem Rev 105:2253–2277. doi: 10.1021/cr0307143 CrossRefPubMedGoogle Scholar
  4. 4.
    Hannemann F, Bichet A, Ewen KM, Bernhardt R (2007) Biochim Biophys Acta 1770:330–344. doi: 10.1016/j.bbagen.2006.07.017 PubMedGoogle Scholar
  5. 5.
    Munro AW, Girvan HM, McLean KJ (2007) Biochim Biophys Acta 1770:345–359. doi: 10.1016/j.bbagen.2006.08.018 PubMedGoogle Scholar
  6. 6.
    Bell SG, Hoskins N, Xu F, Caprotti D, Rao Z, Wong LL (2006) Biochem Biophys Res Commun 342:191–196. doi: 10.1016/j.bbrc.2006.01.133 CrossRefPubMedGoogle Scholar
  7. 7.
    Bell SG, Xu F, Forward I, Bartlam M, Rao Z, Wong L-L (2008) J Mol Biol 383:561–574. doi: 10.1016/j.jmb.2008.08.033 CrossRefPubMedGoogle Scholar
  8. 8.
    Peng Y, Xu F, Bell SG, Wong LL, Rao Z (2007) Acta Cryst F 63:422–425. doi: 10.1107/S1744309107017411 CrossRefGoogle Scholar
  9. 9.
    Xu F, Bell SG, Peng Y, Johnson EO, Bartlam M, Rao Z, Wong LL (2009) Proteins 77:867–880. doi: 10.1002/prot.22510 CrossRefPubMedGoogle Scholar
  10. 10.
    Seo D, Okabe S, Yanase M, Kataoka K, Sakurai T (2009) Biochim Biophys Acta 1794:594–601. doi: 10.1016/j.bbapap.2008.12.014 PubMedGoogle Scholar
  11. 11.
    Peterson JA, Lorence MC, Amarneh B (1990) J Biol Chem 265:6066–6073PubMedGoogle Scholar
  12. 12.
    Westlake AC, Harford-Cross CF, Donovan J, Wong LL (1999) Eur J Biochem 265:929–935. doi: 10.1046/j.1432-1327.1999.00793.x CrossRefPubMedGoogle Scholar
  13. 13.
    Blair DE, Diehl H (1961) Anal Chem 33:867–870. doi: 10.1021/ac60175a016 CrossRefGoogle Scholar
  14. 14.
    Moulis JM, Meyer J (1982) Biochemistry 21:4762–4771. doi: 10.1021/bi00262a037 CrossRefPubMedGoogle Scholar
  15. 15.
    Zheng L, Cash VL, Flint DH, Dean DR (1998) J Biol Chem 273:13264–13272CrossRefPubMedGoogle Scholar
  16. 16.
    Nakamura M, Saeki K, Takahashi Y (1999) J Biochem 126:10–18PubMedGoogle Scholar
  17. 17.
    Lill R, Kispal G (2000) Trends Biochem Sci 25:352–356. doi: S0968-0004(00)01589-9 CrossRefPubMedGoogle Scholar
  18. 18.
    Sainz G, Jakoncic J, Sieker LC, Stojanoff V, Sanishvili N, Asso M, Bertrand P, Armengaud J, Jouanneau Y (2006) J Biol Inorg Chem 11:235–246. doi: 10.1007/s00775-005-0069-2 CrossRefPubMedGoogle Scholar
  19. 19.
    Lambeth JD, Seybert DW, Kamin H (1979) J Biol Chem 254:7255–7264PubMedGoogle Scholar
  20. 20.
    Aoki M, Ishimori K, Morishima I, Wada Y (1998) Inorg Chim Acta 272:80–88. doi: 10.1016/S0020-1693(97)05946-X CrossRefGoogle Scholar
  21. 21.
    Müller A, Müller JJ, Muller YA, Uhlmann H, Bernhardt R, Heinemann U (1998) Structure 6:269–280CrossRefPubMedGoogle Scholar
  22. 22.
    Cupp JR, Vickery LE (1989) J Biol Chem 264:1602–1607PubMedGoogle Scholar
  23. 23.
    Grinberg AV, Hannemann F, Schiffler B, Muller J, Heinemann U, Bernhardt R (2000) Proteins 40:590–612. doi: 10.1002/1097-0134 CrossRefPubMedGoogle Scholar
  24. 24.
    Schiffler B, Bernhardt R (2003) Biochem Biophys Res Commun 312:223–228. doi: 10.1016/j.bbrc.2003.09.214 CrossRefPubMedGoogle Scholar
  25. 25.
    Kakuta Y, Horio T, Takahashi Y, Fukuyama K (2001) Biochemistry 40:11007–11012. doi: 10.1021/bi010544t CrossRefPubMedGoogle Scholar
  26. 26.
    Bell SG, Wong LL (2007) Biochem Biophys Res Commun 360:666–672. doi: 10.1016/j.bbrc.2007.06.119 CrossRefPubMedGoogle Scholar
  27. 27.
    Bell SG, Dale A, Rees NH, Wong L-L (2009) Appl Microbiol Biotechnol. doi: 10.1007/s00253-009-2234-y
  28. 28.
    Davies MD, Qin L, Beck JL, Suslick KS, Koga H, Horiuchi T, Sligar SG (1990) J Am Chem Soc 112:7396–7398. doi: 10.1021/ja00176a050 CrossRefGoogle Scholar
  29. 29.
    Holden M, Mayhew M, Bunk D, Roitberg A, Vilker V (1997) J Biol Chem 272:21720–21725. doi: 10.1074/jbc.272.35.21720 CrossRefPubMedGoogle Scholar
  30. 30.
    Sevrioukova IF, Garcia C, Li H, Bhaskar B, Poulos TL (2003) J Mol Biol 333:377–392. doi: 10.1016/j.jmb.2003.08.028 CrossRefPubMedGoogle Scholar
  31. 31.
    Kuznetsov VY, Blair E, Farmer PJ, Poulos TL, Pifferitti A, Sevrioukova IF (2005) J Biol Chem 280:16135–16142. doi: 10.1074/jbc.M500771200 CrossRefPubMedGoogle Scholar
  32. 32.
    Kuznetsov VY, Poulos TL, Sevrioukova IF (2006) Biochemistry 45:11934–11944. doi: 10.1021/bi0611154 CrossRefPubMedGoogle Scholar
  33. 33.
    Beckert V, Dettmer R, Bernhardt R (1994) J Biol Chem 269:2568–2573PubMedGoogle Scholar
  34. 34.
    Pochapsky TC, Lyons TA, Kazanis S, Arakaki T, Ratnaswamy G (1996) Biochimie 78:723–733CrossRefPubMedGoogle Scholar
  35. 35.
    Davies MD, Sligar SG (1992) Biochemistry 31:11383–11389. doi: 10.1021/bi00161a016 CrossRefPubMedGoogle Scholar
  36. 36.
    Roitberg AE, Holden MJ, Mayhew MP, Kurnikov IV, Beratan DN, Vilker VL (1998) J Am Chem Soc 120:8927–8932. doi: 10.1021/ja9739906 CrossRefGoogle Scholar
  37. 37.
    Shimada H, Nagano S, Ariga Y, Unno M, Egawa T, Hishiki T, Ishimura Y, Masuya F, Obata T, Hori H (1999) J Biol Chem 274:9363–9369CrossRefPubMedGoogle Scholar
  38. 38.
    Hannemann F, Rottmann M, Schiffler B, Zapp J, Bernhardt R (2001) J Biol Chem 276:1369–1375. doi: 10.1074/jbc.M007589200 CrossRefPubMedGoogle Scholar
  39. 39.
    Brandt ME, Vickery LE (1993) J Biol Chem 268:17126–17130PubMedGoogle Scholar

Copyright information

© SBIC 2009

Authors and Affiliations

  • Stephen G. Bell
    • 1
    Email author
  • Feng Xu
    • 2
  • Eachan O. D. Johnson
    • 1
  • Ian M. Forward
    • 1
  • Mark Bartlam
    • 3
  • Zihe Rao
    • 2
    • 3
    Email author
  • Luet-Lok Wong
    • 1
  1. 1.Department of Chemistry, Inorganic Chemistry LaboratoryUniversity of OxfordOxfordUK
  2. 2.Tsinghua-Nankai-IBP Joint Research Group for Structural BiologyTsinghua UniversityBeijingChina
  3. 3.Tianjin Key Laboratory of Protein Science, College of Life SciencesNankai UniversityTianjinChina

Personalised recommendations