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Replacement of tyrosine residues by phenylalanine in cytochrome P450cam alters the formation of Cpd II-like species in reactions with artificial oxidants

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Abstract

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [Spolitak et al. (2005) J Biol Chem 280:20300–20309; (2006) J Inorg Biochem 100:2034–2044] spectrally characterized compound I [ferryl iron plus a porphyrin π-cation radical (FeIV=O/Por·+)], as well as Cpd ES (FeIV=O/Tyr·). In the present studies, we report how the substitutions in Y75F, Y96F, and Y96F/Y75F P450cam variants permit the formation of a species we attribute to Cpd II (FeIV=O) in reactions with peracids and cumene hydroperoxide. These variants produce changes in hydrogen bonding patterns and increased hydrophobicity that affect the ratio of heterolytic to homolytic pathways in reactions with cumene hydroperoxide, resulting in a shift of this ratio from 84/16 for WT to 72/28 for the Y96F/Y75F double mutant. Various ways of generating the Cpd II-like species were explored, and it was possible, especially with the more hydrophobic variants, to generate large fractions of the P450cam variants as Cpd II. The Cpd II-like species is ineffective at hydroxylating camphor, but can be readily reduced by ascorbate (as well as other peroxidase substrates) to ferric P450cam, which could then bind camphor to form the high-spin heme. The difference in the spectral properties of Cpd ES and Cpd II was rationalized as possibly being due to different states of protonation.

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Notes

  1. We also used a double mixing stopped-flow method to test whether the mCPBA could have reacted with the methanol before it was mixed with the P450cam. Methanol and mCPBA were combined in the first mix, and after delays of 50 ms–100 s the mixture was combined with the P450cam. The results were the same regardless of the delay; Cpd II formed and then slowly converted back to the ferric form, as seen in Fig. 4.

Abbreviations

Cpd I:

State of cytochrome that is two equivalents of oxidation greater than the ferric form

Cpd II:

State of cytochrome that is one equivalent of oxidation greater than the ferric form

Cpd ES:

The two-electron-oxidized state of P450 or peroxidases containing both an oxoferryl center [FeIV=O] and either a tryptophanyl or tyrosyl radical, analogous to Cpd ES in cytochrome c peroxidase

CPO:

Chloroperoxidase from Caldariomyces fumago

CumOOH:

Cumene hydroperoxide

HRP:

Horseradish peroxidase

mCPBA:

meta-Chloroperbenzoic acid

PAA:

Peracetic acid

P450:

Cytochrome P450

P450cam:

Cytochrome P450cam isolated from Pseudomonas putida

RFQ-EPR:

Rapid freeze-quench electron paramagnetic resonance spectroscopy

TMPD:

N,N,N,N-tetramethyl-p-phenylenediamine

Y75F, Y96F, and Y75F/Y96F:

Single and double mutants of P450cam

References

  1. Ortiz de Montellano PR (ed) (2005) Cytochrome P450: structure, mechanism and biochemistry, 3rd edn. Kluwer/Plenum, New York

  2. Hayaishi O (1974) Molecular mechanisms of oxygen activation. Academic, New York

  3. Groves JT (2003) Proc Natl Acad Sci USA 100:3569–3574

    Article  PubMed  CAS  Google Scholar 

  4. Coon MJ (2003) Biochem Biophys Res Commun 312:163–168

    Article  PubMed  CAS  Google Scholar 

  5. Davydov R, Makris TM, Koffman V, Werst DE, Sligar SG, Hoffman BM (2001) J Am Chem Soc 123:1403–1415

    Article  PubMed  CAS  Google Scholar 

  6. Sligar SG, Makris TM, Denisov IG (2005) Biochem Biophys Res Commun 338:346–354

    Article  PubMed  CAS  Google Scholar 

  7. Newcomb M, Chandrasena RE (2005) Biochem Biophys Res Commun 338:394–403

    Article  PubMed  CAS  Google Scholar 

  8. Nam W, Ryu YO, Song WJ (2004) J Biol Inorg Chem 9:654–660

    Article  PubMed  CAS  Google Scholar 

  9. McLain J, Lee J, Groves JT (2000) In: Meunier B (ed) Biomimetic oxidations catalyzed by transition metal complexes. Imperial College Press, London, pp 91–170

  10. Egawa T, Shimada H, Ishimura Y (1994) Biochem Biophys Res Commun 201:1464–1469

    Article  PubMed  CAS  Google Scholar 

  11. Kellner DG, Hung SC, Weiss KE, Sligar SG (2002) J Biol Chem 277:9641–9644

    Article  PubMed  CAS  Google Scholar 

  12. Sono M, Roach MP, Coulter ED, Dawson JH (1996) Chem Rev 96:2841–2887

    Article  PubMed  CAS  Google Scholar 

  13. Isaac IS, Dawson JH (1999) In: Ballou DP (ed) Essays in biochemistry. Portland, London, UK, pp 51–69

  14. Spolitak T, Dawson JH, Ballou DP (2005) J Biol Chem 280:20300–20309

    Article  PubMed  CAS  Google Scholar 

  15. Spolitak T, Dawson JH, Ballou DP (2006) J Inorg Biochem 100:2034–2044

    Article  PubMed  CAS  Google Scholar 

  16. Schünemann V, Lendzian F, Jung C, Contzen J, Barra AL, Sligar SG, Trautwein AX (2004) J Biol Chem 279:10919–10930

    Article  PubMed  CAS  Google Scholar 

  17. Schünemann V, Jung C, Terner J, Trautwein AX, Weiss R (2002) J Inorg Biochem 91:586–596

    Article  PubMed  Google Scholar 

  18. Raner GM, Thompson JI, Haddy A, Tangham V, Bynum N, Ramachandra Reddy G, Ballou DP, Dawson JH (2006) J Inorg Biochem 100:2045–2053

    Article  PubMed  CAS  Google Scholar 

  19. Sato H, Guengerich FP (2000) J Am Chem Soc 122:8099–8100

    Article  CAS  Google Scholar 

  20. Daiber A, Herold S, Schoneich C, Namgaladze D, Peterson JA, Ullrich V (2000) Eur J Biochem 267:6729–6739

    PubMed  CAS  Google Scholar 

  21. Daiber A, Schoneich C, Schmidt P, Jung C, Ullrich V (2000) J Inorg Biochem 81:213–220

    Article  PubMed  CAS  Google Scholar 

  22. Newcomb M, Zhang R, Chandrasena RE, Halgrimson JA, Horner JH, Makris TM, Sligar SG (2006) J Am Chem Soc 128:4580–4581

    Article  PubMed  CAS  Google Scholar 

  23. Mehl M, Daiber A, Herold S, Shoun H, Ullrich V. (1999) Nitric Oxide Biol Chem 3:142–152

    Article  CAS  Google Scholar 

  24. Behan RK, Hoffart LM, Stone KL, Krebs C, Green MT (2007) J Am Chem Soc 129:5855–5859

    Article  PubMed  CAS  Google Scholar 

  25. Rodriguez-Lopez JN, Smith A, Thorneley RNF (1996) J Biol Inorg Chem 1:136–142

    Article  CAS  Google Scholar 

  26. Matsui T, Nagano S, Ishimori K, Watanabe Y, Morishima I (1996) Biochemistry 35:13118–13124

    Article  PubMed  CAS  Google Scholar 

  27. Finzel BC, Poulos TL, Kraut J (1984) J Biol Chem 259:13027–13036

    PubMed  CAS  Google Scholar 

  28. Newmyer SL, Ortiz de Montellano PR (1996) J Biol Chem 271:14891–14896

    Article  PubMed  CAS  Google Scholar 

  29. Choudhury K, Sundaramoorthy M, Hickman A, Yonetani T, Woehl E, Dunn MF, Poulos TL (1994) J Biol Chem 269:20239–20249

    PubMed  CAS  Google Scholar 

  30. Tanaka M, Ishimori K, Mukai M, Kitagawa T, Morishima I (1997) Biochemistry 36:9889–9898

    Article  PubMed  CAS  Google Scholar 

  31. Unger BP, Gunsalus IC, Sligar SG (1986) J Biol Chem 261:1158–1163

    PubMed  CAS  Google Scholar 

  32. Taylor JW, Ott J, Eckstein F (1985) Nucleic Acids Res 13:8765–8785

    Article  PubMed  CAS  Google Scholar 

  33. Atkins WM, Sligar SG (1988) J Biol Chem 263:18842–18849

    PubMed  CAS  Google Scholar 

  34. Egawa T, Ogura T, Makino R, Ishimura Y, Kitagawa T (1991) J Biol Chem 266:10246–10248

    PubMed  CAS  Google Scholar 

  35. Cotton ML, Dunford HB, Raycheba JM (1973) Can J Biochem 51:627–631

    Article  PubMed  CAS  Google Scholar 

  36. Bevington PR (1996) In: Data reduction and error analysis for the physical sciences. McGraw-Hill Inc., New York, pp 235–242

  37. Jakopitsch C, Auer M, Ivancich A, Ruker F, Furtmuller PG, Obinger C (2003) J Biol Chem 278:20185–20191

    Article  PubMed  CAS  Google Scholar 

  38. Yoshioka S, Takahashi S, Ishimori K, Morishima I (2000) J Inorg Biochem 81:141–151

    Article  PubMed  CAS  Google Scholar 

  39. Prasad S, Mazumdar S, Mitra S (2000) FEBS Lett 477:157–160

    Article  PubMed  CAS  Google Scholar 

  40. Griffin BW, Peterson JA (1972) Biochemistry 11:4740–4746

    Article  PubMed  CAS  Google Scholar 

  41. Behan RK, Hoffart LM, Stone KL, Krebs C, Green MT (2006) J Am Chem Soc 128:11471–11474

    Article  PubMed  CAS  Google Scholar 

  42. Behan RK, Green MT (2006) J Inorg Biochem 100:448–459

    Article  PubMed  CAS  Google Scholar 

  43. Balasubramanian PN, Lee RW, Bruice TC (1989) J Am Chem Soc 111:8714–8721

    Article  CAS  Google Scholar 

  44. Murata K, Panicucci R, Gopinath E, Bruice TC (1990) J Am Chem Soc 112:6072–6083

    Article  CAS  Google Scholar 

  45. Matsui T, Ozaki S, Watanabe Y (1999) J Am Chem Soc 121:9952–9957

    Article  CAS  Google Scholar 

  46. Matsui T, Ozaki S, Watanabe Y (1997) J Biol Chem 272:32735–32738

    Article  PubMed  CAS  Google Scholar 

  47. Dawson JH, Andersson LA, Sono M (1983) J Biol Chem 258:13637–13645

    PubMed  CAS  Google Scholar 

  48. Watanabe Y, Nakajima H, Ueno T (2007) Acc Chem Res 40:554–562

    Article  PubMed  CAS  Google Scholar 

  49. Altun A, Shaik S, Thiel W (2007) J Am Chem Soc 129:8978–8987

    Article  PubMed  CAS  Google Scholar 

  50. Nam W, Park S-E, Lim IK, Lim MH, Hong J, Kim J (2003) J Am Chem Soc 125:14674–14675

    Article  PubMed  CAS  Google Scholar 

  51. Groves JT (2005) In: Ortiz de Montelano PR (ed) Cytochrome P450: structure, mechanism, and biochemistry, 3rd edn. Kluwer/Plenum, New York, pp 1–43

  52. Shaik S, Kumar D, de Visser SP, Altun A, Thiel W (2005) Chem Rev 105:2279–2328

    Article  PubMed  CAS  Google Scholar 

  53. Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, Benson DE, Sweet BM, Ringe D, Petsko GA, Sligar SG (2000) Science 287:1615–1622

    Article  PubMed  CAS  Google Scholar 

  54. Schoneboom JC, Cohen S, Lin H, Shaik S, Thiel W (2004) J Am Chem Soc 126:4017–4034

    Article  PubMed  CAS  Google Scholar 

  55. Green MT, Dawson JH, Gray HB (2004) Science 304:1653–1656

    Article  PubMed  CAS  Google Scholar 

  56. Jung C, Schunemann V, Lendzian F (2005) Biochem Biophys Res Commun 338:355–364

    Article  PubMed  CAS  Google Scholar 

  57. Silaghi-Dumitrescu R, Reeder BJ, Nicholls P, Cooper CE, Wilson MT (2007) Biochem J 403:391–395

    Article  PubMed  CAS  Google Scholar 

  58. Stone KL, Behan RK, Green MT (2006) Proc Natl Acad Sci USA 103:12307–12310

    Article  PubMed  CAS  Google Scholar 

  59. Terner J, Palaniappan V, Gold A, Weiss R, Fitzgerald MM, Sullivan AM, Hosten CM (2006) J Inorg Biochem 100:480–501

    Article  PubMed  CAS  Google Scholar 

  60. Derat E, Shaik S (2006) J Am Chem Soc 128:8185–8198

    Article  PubMed  CAS  Google Scholar 

  61. Groves JT (2006) J Inorg Biochem 100:434–447

    Article  PubMed  CAS  Google Scholar 

  62. Chance M, Powers L, Poulos T, Chance B (1986) Biochemistry 25:1266–1270

    Article  PubMed  CAS  Google Scholar 

  63. Hashimoto S, Teraoka J, Inubushi T, Yonetani T, Kitagawa T (1986) J Biol Chem 261:11110–11118

    PubMed  CAS  Google Scholar 

  64. Stone KL, Hoffart LM, Behan RK, Krebs C, Green MT (2006) J Am Chem Soc 128:6147–6153

    Article  PubMed  CAS  Google Scholar 

  65. Glascock MC, Ballou DP, Dawson JH (2005) J Biol Chem 280:42134–42141

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was support provided by grants from the National Institutes of Health, GM20877 to D.P.B. and GM26730 to J.H.D. We are very grateful to Dr. J. Windak, Supervisor of Instrumental Services, Chemistry Department of the University of Michigan, for his assistance in setting up GC–MS analysis. We would like to acknowledge helpful reviewer comments.

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Authors and Affiliations

  1. Department of Biological Chemistry, Medical School, University of Michigan, Ann Arbor, MI, 48109-0606, USA

    Tatyana Spolitak & David P. Ballou

  2. Department of Chemistry and Biochemistry and the School of Medicine, University of South Carolina, Columbia, SC, 29208, USA

    John H. Dawson

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  1. Tatyana Spolitak
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  2. John H. Dawson
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  3. David P. Ballou
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Correspondence to David P. Ballou.

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Spolitak, T., Dawson, J.H. & Ballou, D.P. Replacement of tyrosine residues by phenylalanine in cytochrome P450cam alters the formation of Cpd II-like species in reactions with artificial oxidants. J Biol Inorg Chem 13, 599–611 (2008). https://doi.org/10.1007/s00775-008-0348-9

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