Critical Review Histidine and not Tyrosine is Required for the Peroxide-induced Formation of Haem to Protein Cross-linked Myoglobin Brandon J. Reeder 1 , Francesca Cutruzzola` 2 , Maria Giulia Bigotti 2 , Nicholas J. Watmough 3 and Michael T. Wilson 1 1 Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, UK 2 University of Rome La Sapienza, Department of Biochemical Sciences, Rome, Italy 3 School of Biological Sciences, University of East Anglia, Norwich, UK Summary Peroxide-induced oxidative modifications of haem proteins such as myoglobin and haemoglobin can lead to the formation of a covalent bond between the haem and globin. These haem to protein cross-linked forms of myoglobin and haemoglobin are cytotoxic and have been identified in pathological conditions in vivo. An under- standing of the mechanism of haem to protein cross-link formation could provide important information on the mechanisms of the oxidative processes that lead to pathological complications asso- ciated with the formation of these altered myoglobins and haemoglobins. We have re-examined the mechanism of the formation of haem to protein cross-link to test the previously reported hypothesis that the haem forms a covalent bond to the protein via the tyrosine 103 residue (Catalano, C. E., Choe, Y. S., Ortiz de Montellano, P. R., J. Biol. Chem. 1989, 10534 – 10541). Comparison of native horse myoglobin, recombinant sperm whale myoglobin and Tyr 103 ! Phe sperm whale mutant shows that, contrary to the previously proposed mechanism of haem to protein cross-link formation, the absence of tyrosine 103 has no impact on the formation of haem to protein cross-links. In contrast, we have found that engineered myoglobins that lack the distal histidine residue either cannot generate haem to protein cross-links or show greatly suppressed levels of modified protein. Moreover, addition of a distal histidine to myoglobin from Aplysia limacina, that naturally lacks this histidine, restores the haem protein’s capacity to generate haem to protein cross-links. The distal histidine is, therefore, vital for the formation of haem to protein cross-link and we explore this outcome. IUBMB Life, 59: 477–489, 2007 Keywords Myoglobin; histidine; tyrosine; peroxide; aplysia; ferryl. Abbreviations Hb, Haemoglobin; Hb-X, Haem to protein cross- linked haemoglobin; HH, Horse Heart; Mb, Myoglobin; Mb-X, Haem to protein cross-linked myoglobin; SW, Sperm Whale. INTRODUCTION Myoglobin (Mb) is a respiratory haem protein that carries oxygen and now appears to be involved in NO binding. However, Mb can also exhibit a rogue enzymatic activity in which it can react with peroxides and, through free radical chemistry, initiate a complex series of lipid oxidation reactions. The pseudo-peroxidase activity of Mb leads to the oxidation of ferrous or ferric Mb to the ferryl state and, in the case of ferric Mb, the formation of ferryl plus a protein-based radical. Numerous studies have examined the characteristics of these peroxide-induced protein radicals by spin trapping and direct EPR spectroscopy, using both native and mutant Mb species (1–4). These studies have identified protein radicals on a variety of residues, including a tyrosine residue close to the haem (Tyr 103 , sperm whale) and a tyrosine close to the protein surface (Tyr 151 , sperm whale). The latter of these tyrosine radicals has been shown to be involved in protein- protein cross-linking of Mb (3, 5, 6). Tryptophan has also been identified to harbour a radical (Trp 14 ), which appears as a peroxyl radical in the presence of oxygen (5, 7). The concentration of total protein radical detected is very low in comparison with the concentration of myoglobin. The discrepancy in this stoichiometry of ferryl to radical is attributed to a fast dispersion of the radical, which can result in oxidative modifications to the protein, haem, or exogenous substrates (8 – 12). Under acidic conditions the protein radical can react with the protonated form of the oxo-ferryl haem (Fe 4þ -OH 7 ), resulting in the formation of a covalent bond between the haem porphyrin ring and the protein (13). This haem to protein cross-linked form of Mb (Mb-X) has Received 15 December 2006; accepted 18 December 2006 Address correspondence to: Dr Brandon J. Reeder, Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, UK. Tel: þ1 (0)1206 872119. Fax: þ1 (0)1206 872592. E-mail: reedb@essex.ac.uk IUBMB Life, 59(8 – 9): 477 – 489, August – September 2007 ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2007 IUBMB DOI: 10.1080/15216540601178083