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Article

Autocatalytic Formation of a Covalent Link between Tryptophan 41 and the Heme in Ascorbate Peroxidase^†

Supporting Info

Zoi Pipirou,^‡ Andrew R. Bottrill,^§ Clive M. Metcalfe,^‡ Sharad C. Mistry,^§ Sandip K. Badyal,^‡ Bernard J. Rawlings,^‡ and Emma Lloyd Raven*^‡

Department of Chemistry, University of Leicester, University Road, Leicester LE1 7RH, England, and Protein and Nucleic Acid Chemistry Laboratory, Hodgkin Building, University of Leicester, Lancaster Road, Leicester LE1 9HN, U.K.

Biochemistry, 2007, 46 (8), pp 2174–2180

DOI: 10.1021/bi062274q

Publication Date (Web): January 31, 2007

†

This work was supported by grants from The Leverhulme Trust and BBSRC (Grants RF/RFG/2005/0299 and BB/C001184/1 to E.L.R.), the EPSRC (studentship to Z.P.), and BBSRC (studentship to S.K.B.).

‡

Department of Chemistry, University of Leicester

Protein and Nucleic Acid Chemistry Laboratory, University of Leicester.

To whom correspondence should be addressed. Telephone: +44 (0)116 229 7047. Fax: +44 (0)116 252 2789. E-mail: emma.raven@ le.ac.uk.

Abstract

Electronic spectroscopy, HPLC analyses, and mass spectrometry (MALDI-TOF and MS/MS) have been used to show that a covalent link from the heme to the distal Trp41 can occur on exposure of ascorbate peroxidase (APX) to H₂O₂ under noncatalytic conditions. Parallel analyses with the W41A variant and with APX reconstituted with deuteroheme clearly indicate that the covalent link does not form in the absence of either Trp41 or the heme vinyl groups. The presence of substrate also precludes formation of the link. Formation of a protein radical at Trp41 is implicated, in a reaction mechanism that is analogous to that proposed [Ghiladi, R. A., et al. (2005) Biochemistry 44, 15093−15105] for formation of a covalent Trp-Tyr-Met link in the closely related catalase peroxidase (KatG) enzymes. Collectively, the data suggest that radical formation at the distal tryptophan position is not an exclusive feature of the KatG enzymes and may be used more widely across other members of the class I heme peroxidase family.

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