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LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2


LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2

Kathiresan, Meena and English, Ann M. ORCID: https://orcid.org/0000-0002-3696-7710 (2017) LC-MS/MS suggests that hole hopping in cytochrome c peroxidase protects its heme from oxidative modification by excess H2O2. Chemical Science, 8 (2). pp. 1152-1162. ISSN 2041-6520

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Official URL: http://dx.doi.org/10.1039/c6sc03125k


We recently reported that cytochrome c peroxidase (Ccp1) functions as a H2O2 sensor protein when H2O2 levels rise in respiring yeast. The availability of its reducing substrate, ferrocytochrome c (CycII), determines whether Ccp1 acts as a H2O2 sensor or peroxidase. For H2O2 to serve as a signal it must modify its receptor so we employed high-performance LC-MS/MS to investigate in detail the oxidation of Ccp1 by 1, 5 and 10 M eq. of H2O2 in the absence of CycII to prevent peroxidase activity. We observe strictly heme-mediated oxidation, implicating sequential cycles of binding and reduction of H2O2 at Ccp1's heme. This results in the incorporation of ∼20 oxygen atoms predominantly at methionine and tryptophan residues. Extensive intramolecular dityrosine crosslinking involving neighboring residues was uncovered by LC-MS/MS sequencing of the crosslinked peptides. The proximal heme ligand, H175, is converted to oxo-histidine, which labilizes the heme but irreversible heme oxidation is avoided by hole hopping to the polypeptide until oxidation of the catalytic distal H52 in Ccp1 treated with 10 M eq. of H2O2 shuts down heterolytic cleavage of H2O2 at the heme. Mapping of the 24 oxidized residues in Ccp1 reveals that hole hopping from the heme is directed to three polypeptide zones rich in redox-active residues. This unprecedented analysis unveils the remarkable capacity of a polypeptide to direct hole hopping away from its active site, consistent with heme labilization being a key outcome of Ccp1-mediated H2O2 signaling. LC-MS/MS identification of the oxidized residues also exposes the bias of electron paramagnetic resonance (EPR) detection toward transient radicals with low O2 reactivity.

Divisions:Concordia University > Faculty of Arts and Science > Chemistry and Biochemistry
Item Type:Article
Authors:Kathiresan, Meena and English, Ann M.
Journal or Publication:Chemical Science
  • Funding for this research was provided by: Natural Sciences and Engineering Research Council of Canada (Discovery Grant)
Digital Object Identifier (DOI):10.1039/c6sc03125k
ID Code:985119
Deposited On:28 May 2019 14:14
Last Modified:29 May 2019 12:44
Additional Information:Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c6sc03125k http://www.rsc.org/suppdata/c6/sc/c6sc03125k/c6sc03125k1.pdf


1. D. A. Svistunenko, M. T. Wilson and C. E. Cooper, Biochim. Biophys. Acta, Bioenerg., 2004, 1655, 372–380.
2. J. Stubbe and W. A. van Der Donk, Chem. Rev., 1998, 98, 705–762.
3. E. C. Minnihan, D. G. Nocera and J. Stubbe, Acc. Chem. Res., 2013, 46, 2524–2535.
4. A.-L. Tsai, R. J. Kulmacz and G. Palmer, J. Biol. Chem., 1995,270, 10503–10508.
5. E. T. Yukl, H. R. Williamson, L. Higgins, V. L. Davidson and C. M. Wilmot, Biochemistry, 2013, 52, 9447–9455.
6. H. B. Gray and J. R. Winkler, Proc. Natl. Acad. Sci. U. S. A.,2015, 112, 10920–10925.
7. A. M. English and G. Tsaprailis, Adv. Inorg. Chem., 1995, 43, 9–125.
8. A. N. Volkov, P. Nicholls and J. A. R. Worrall, Biochim. Biophys. Acta, Bioenerg., 2011, 1807, 1482–1503.
9. M. Sivaraja, D. B. Goodin, M. Smith and B. M. Hoffman, Science, 1989, 245, 738–740.
10. J. E. Erman and L. B. Vitello, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 2002, 1597, 193–220.
11. M. A. Miller, L. B. Vitello and J. E. Erman, Biochemistry, 1995, 34, 12048–12058.
12. D. Martins, M. Kathiresan and A. M. English, Free Radical Biol. Med., 2013, 65, 541–551.
13. H. Jiang and A. M. English, J. Inorg. Biochem., 2006, 100,1996–2008.
14. M. Kathiresan, D. Martins and A. M. English, Proc. Natl.Acad. Sci. U. S. A., 2014, 111, 17468–17473.
15. J. E. Erman and T. Yonetani, Biochim. Biophys. Acta, Protein Struct., 1975, 393, 350–357.
16. H. Hori and T. Yonetani, J. Biol. Chem., 1985, 260, 349–355.
17. L. A. Fishel, M. F. Farnum, J. M. Mauro, M. A. Miller, J. Kraut,Y. Liu, X. L. Tan and C. P. Scholes, Biochemistry, 1991, 30,1986–1996.
18. D. B. Goodin, A. G. Mauk and M. Smith, Proc. Natl. Acad. Sci. U. S. A., 1986, 83, 1295–1299.
19. R. A. Musah and D. B. Goodin, Biochemistry, 1997, 36, 11665–11674.
20. A. Ivancich, P. Dorlet, D. B. Goodin and S. Un, J. Am. Chem. Soc., 2001, 123, 5050–5058.
21. K. D. Miner, T. D. Pfister, P. Hosseinzadeh, N. Karaduman, L. J. Donald, P. C. Loewen, Y. Lu and A. Ivancich,
Biochemistry, 2014, 53, 3781–3789.
22. H. Zhang, S. He and A. G. Mauk, Biochemistry, 2002, 41, 13507–13513.
23. G. Tsaprailis and A. M. English, J. Biol. Inorg. Chem., 2003, 8, 248–255.
24. A. Filosa and A. M. English, J. Biol. Chem., 2001, 276, 21022–21027.
25. C. W. Fenwick and A. M. English, J. Am. Chem. Soc., 1996,118, 12236–12237.
26. P. J. Wright and A. M. English, J. Am. Chem. Soc., 2003, 125, 8655–8665.
27. I. Dalle-Donne, A. Scaloni, D. Giustarini, E. Cavarra, G. Tell, G. Lungarella, R. Colombo, R. Rossi and A. Milzani, Mass Spectrom. Rev., 2005, 24, 55–99.
28. C. Schöneich and V. S. Sharov, Free Radical Biol. Med., 2006, 41, 1507–1520.
29. O. Chárvatová, B. L. Foley, M. W. Bern, J. S. Sharp, R. Orlando and R. J. Woods, J. Am. Soc. Mass Spectrom., 2008, 19, 1692–1705.
30. L. Konermann, B. Stocks, Y. Pan and X. Tong, Mass Spectrom. Rev., 2010, 29, 651–667.
31. M. Krauss and D. R. Garner, J. Phys. Chem., 1993, 97, 831–836.
32. G. Tsaprailis and A. M. English, Can. J. Chem., 1996, 74, 2250–2257.
33. T. Fox, G. Tsaprailis and A. M. English, Biochemistry, 1994, 33, 186–191.
34. B. D. Spangler and J. E. Erman, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1986, 872, 155–157.
35. T. D. Pfister, A. J. Gengenbach, S. Syn and Y. Lu, Biochemistry, 2001, 40, 14942–14951.
36. B. S. Berlett and E. R. Stadtman, J. Biol. Chem., 1997, 272, 20313–20316.
37. M. F. Beal, Free Radical Biol. Med., 2002, 32, 797–803.
38. R. L. Levine and E. R. Stadtman, Exp. Gerontol., 2001, 36, 1495–1502.
39. I. M. Møller, A. Rogowska-Wrzesinska and R. S. P. Rao, J. Proteomics, 2011, 74, 2228–2242.
40. E. A. Veal, A. M. Day and B. A. Morgan, Mol. Cell, 2007, 26, 1–14.
41. H. J. Forman, M. Maiorino and F. Ursini, Biochemistry, 2010, 49, 835–842.
42. R. E. Childs and W. G. Bardsley, Biochem. J., 1975, 145, 93–103.
43. A. R. Jones, J. A. Siepen, S. J. Hubbard and N. W. Paton, Proteomics, 2009, 9, 1220–1229.
44. W. Zhu, J. W. Smith and C.-M. Huang, J. Biomed. Biotechnol., 2010, 2010, 1–6.
45. D. A. Malencik, J. F. Sprouse, C. A. Swanson and S. R. Anderson, Anal. Biochem.,1996, 242, 202–213.
46. K. Kim and J. E. Erman, Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol., 1988, 954, 95–107.
47. T. Yonetani, J. Biol. Chem., 1967, 242, 5008–5013.
48. J. E. Erman, L. B. Vitello, M. A. Miller, A. Shaw, K. A. Brown and J. Kraut, Biochemistry, 1993, 32, 9798–9806.
49. W. T. Dixon and D. Murphy, J. Chem. Soc., Faraday Trans. 2,1976, 72, 1221–1230.
50. A. Harriman, J. Phys Chem., 1987, 91, 6102–6104.
51. M. R. DeFelippis, C. P. Murthy, M. Faraggi and M. H. Klapper, Biochemistry, 1989, 28, 4847–4853.
52. M. A. Yu, T. Egawa, K. Shinzawa-Itoh, S. Yoshikawa, S.-R. Yeh, D. L. Rousseau and G. J. Gerfen, Biochim.
Biophys. Acta, Bioenerg., 2011, 1807, 1295–1304.
53. E. P. L. Hunter, M. F. Desrosiers and M. G. Simic, Free Radical Biol. Med., 1989, 6, 581–585.
54. S. V. Jovanovic, S. Steenken and M. G. Simic, J. Phys. Chem.,1991, 95, 684–687.
55. D. A. Svistunenko, Biochim. Biophys. Acta, Bioenerg., 2005,1707, 127–155.
56. L. P. Candeias, P. Wardman and R. P. Mason, Biophys. Chem., 1997, 67, 229–237.
57. S. Carballal, B. Alvarez, L. Turell, H. Botti, B. A. Freeman and R. Radi, Amino Acids, 2007, 32, 543–551.
58. M. D. Sevilla, D. Becker and M. Yan, Int. J. Radiat. Biol., 1990, 57, 65–81.
59. M. D. Sevilla, M. Yan and D. Becker, Biochem. Biophys. Res. Commun., 1988, 155, 405–410.
60. J. Mönig, K.-D. Asmus, L. G. Forni and R. L. Willson, Int. J. Radiat. Biol. Relat. Stud. Phys., Chem. Med., 1987, 52, 589–602.
61. S. Navaratnam and B. J. Parsons, J. Chem. Soc., Faraday Trans., 1998, 94, 2577–2581.
62. M. R. Gunther, J. A. Peters and M. K. Sivaneri, J. Biol. Chem.,2002, 277, 9160–9166.
63. C. Schöneich, Arch. Biochem. Biophys., 2002, 397, 370–376.
64. M. J. Davies, Biochem. J., 2016, 473, 805–825.
65. R. L. Levine, J. Moskovitz and E. R. Stadtman, IUBMB Life, 2000, 50, 301–307.
66. C. Schöneich, Biochim. Biophys. Acta, Proteins Proteomics, 2005, 1703, 111–119.
67. Z. Ma, H. R. Williamson and V. L. Davidson, Biochem. J., 2016, 473, 1769–1775.
68. L. Josimović, I. Janković and S. V. Jovanović, Radiat. Phys. Chem., 1993, 41, 835–841.
69. G. F. Oxenkrug, Isr. J. Psychiatry Relat. Sci., 2011, 47, 56–63.
70. C. A. Bonagura, B. Bhaskar, H. Shimizu, H. Li, M. Sundaramoorthy, D. E. McRee, D. B. Goodin and
T. L. Poulos, Biochemistry, 2003, 42, 5600–5608.
71. J. E. Erman, L. B. Vitello, J. M. Mauro and J. Kraut, Biochemistry, 1989, 28, 7992–7995.
72. C. C. Valley, A. Cembran, J. D. Perlmutter, A. K. Lewis, N. P. Labello, J. Gao and J. N. Sachs, J. Biol. Chem., 2012,
287, 34979–34991.
73. D. B. Goodin and D. E. McRee, Biochemistry, 1993, 32, 3313–3324.
74. K. Uchida and S. Kawakishi, J. Biol. Chem., 1994, 269, 2405–2410.
75. T. Kurahashi, A. Miyazaki, S. Suwan and M. Isobe, J. Am. Chem. Soc., 2001, 123, 9268–9278.
76. C. S. Maria, E. Revilla, A. Ayala, C. R. de la Cruz and A. Machado, FEBS Lett., 1995, 374, 85–88.
77. D. Martins and A. M. English, Redox Biol., 2014, 2, 632–639.
78. K. Uchida and S. Kawakishi, FEBS Lett., 1993, 332, 208–210.
79. R. P. Pesavento and W. A. van Der Donk, Adv. Protein Chem., 2001, 58, 317–385.
80. E. J. Murphy, C. L. Metcalfe, C. Nnamchi, P. C. E. Moody and E. L. Raven, FEBS J., 2012, 279, 1632–1639.
81. G. Jeschke, Biochim. Biophys. Acta, Bioenerg., 2005, 1707, 91–102.
82. C. Bernini, E. Arezzini, R. Basosi and A. Sinicropi, J. Phys. Chem. B, 2014, 118, 9525–9537.
83. H. Hörtner, G. Ammerer, E. Hartter, B. Hamilton, J. Rytka, T. Bilinski and H. Ruis, Eur. J. Biochem., 1982, 128, 179-184.
84. A. A. Sels and C. Cocriamont, Biochem. Biophys. Res.Commun.,1968, 32, 192–198.
85. L. Djavadi-Ohaniance, Y. Rudin and G. Schatz, J. Biol. Chem., 1978, 253, 4402–4407.
86. J. Kaput, M. C. Brandriss and T. Prussak-Wieckowska, J. Cell Biol., 1989, 109, 101–112.
87. T. P. Barrows and T. L. Poulos, Biochemistry, 2005, 44, 14062–14068.
88. Z. Pipirou, A. R. Bottrill, C. M. Metcalfe, S. C. Mistry, S. K. Badyal, B. J. Rawlings and E. L. Raven, Biochemistry, 2007, 46, 2174–2180.
89. Z. Pipirou, V. Guallar, J. Basran, C. L. Metcalfe, E. J. Murphy, A. R. Bottrill, S. C. Mistry and E. L. Raven, Biochemistry, 2009, 48, 3593–3599.
90. M. Kathiresan and A. M. English, Metallomics, 2016, 8, 434–443.
91. K. Inoue, C. Garner, B. L. Ackermann, T. Oe and I. A. Blair, Rapid Commun. Mass Spectrom., 2006, 20, 911–918.
92. J. M. Gebicki, T. Nauser, A. Domazou, D. Steinmann, P. L. Bounds and W. H. Koppenol, Amino Acids, 2010, 39,
93. J.-W. Lee and J. D. Helmann, Nature, 2006, 440, 363–367.
94. D. A. K. Traoré, A. El Ghazouani, L. Jacquamet, F. Borel, J.-L. Ferrer, D. Lascoux, J.-L. Ravanat, M. Jaquinod,
G. Blondin, C. Caux-Thang, V. Duarte and J.-M. Latour, Nat. Chem. Biol., 2009, 5, 53–59.
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