Characteristics and Mechanism of Formation of Peroxide-Induced Heme to Protein Cross-Linking in Myoglobin ² Brandon J. Reeder,* ,‡ Dimitri A. Svistunenko, ‡ Martyn A. Sharpe, § and Michael T. Wilson ‡ Department of Biological Sciences, Central Campus, UniVersity of Essex, WiVenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom, and Institute of Neurology, UniVersity of London, Queens Square, London WC1N 3BG, United Kingdom ReceiVed June 26, 2001 ABSTRACT: At acidic pH values heme-protein cross-linked myoglobin (Mb-H) forms as a product of a peroxide-induced ferric-ferryl redox cycle. There is evidence that this molecule acts as a marker for heme-protein-induced oxidative stress in vivo and may exacerbate the severity of oxidative damage due to its enhanced prooxidant and pseudoperoxidatic activities. Therefore, an understanding of its properties and mechanism of formation may be important in understanding the association between heme-proteins and oxidative stress. Although the mechanism of formation of heme-protein cross-linked myoglobin is thought to involve a protein radical (possibly a tyrosine) and the ferryl heme, we show that this hypothesis needs revising. We provide evidence that in addition to a protein-based radical the protonated form of the oxoferryl heme, known to be highly reactive and radical-like in nature, is required to initiate cross-linking. This revised mechanism involves radical/radical termination rather than attack of a single radical onto the porphyrin ring. This proposal better explains the pH dependence of cross-linking and may, in part, explain the therapeutic effectiveness of increasing the pH on myoglobin-induced oxidative stress, e.g., therapy for rhabdomyolysis-associated renal dysfunction. Recently, interest has grown regarding the implications for the formation, in vivo, of the heme-protein cross-link species (Mb-H) 1 that results from the reaction of myoglobin with peroxides. Early studies of the reaction between Mb and H 2 O 2 (1-6) showed that this resulted in the production of two derivatives of myoglobin, identifiable from visual inspection by their distinctive colors. A red species is transiently formed from metMb and H 2 O 2 at high pH values. This red species was interpreted to be the ferryl oxidation state of Mb ([Fe 4+ dO 2- ] 2+ )(1), an assignment later con- firmed (5). When metMb reacts with H 2 O 2 at acidic pH values, a stable green species is formed. This green com- pound was tentatively identified as a species formed from substitution on the porphyrin ring without ring fission (1). However, the specific nature of the structures were unknown. Fox et al. noted in 1974 that a proportion of the peroxide- induced green myoglobin species was resistant to acid- solvent extraction, a method that removes the heme group from the protein by disrupting the iron-histidine ligation. Therefore, the green species was identified as a heme group covalently bound to the protein (7). It was not until Catalano et al. examined the green compound in 1989 that this covalent association was confirmed (8). The mechanism of formation of this heme-protein cross-linked species (Mb- H) was conjectured to involve the second oxidizing equiva- lent of hydrogen peroxide forming, among other things, a tyrosine radical that reacts with the heme periphery to form a covalent bond. This form of cross-linking is specific to peroxide-induced ferric-ferryl redox cycling and may be important in the mechanisms of oxidative stress in vivo. Cross-linking the heme to the protein can also be induced by BrCCl 3 but, in this case, covalently linking the heme via the proximal histidine (9-11). Formation of Mb-H is a specific marker for peroxide interaction with Mb as it is stable both in vitro (7, 8) and in vivo (12); it is, therefore, a potentially useful marker for selective oxidation processes. Recent studies of rhabdomy- olysis using an animal model have identified Mb-H excreted in the urine of the animals (rats) used (13). Human patients suffering from rhabdomyolysis also excrete measurable concentrations of Mb-H in their urine (12). Significantly, Mb-H itself is reported to possess potent peroxidative activity. Investigations into the toxicological effects of Mb-H by Osawa and co-workers have shown Mb-H to be a far more powerful oxidant than native Mb. Mb-H can increase NADH oxidation 32-fold (14), increase human fibroblast cell death 5-fold (15), and enhance LDL oxidation (measured by formation of thiobarbituric acid reactive substances, TBARS) 5-fold (16) with respect to native Mb. The accumulated evidence points to Mb-H being a potentially important species, a study of which may advance understanding of the nature of oxidative stress involving respiratory heme-protein. Despite this, the experimental data regarding the properties of Mb-H are limited. The mech- anism of formation described by Catalano et al. is, as the authors state, “not unambiguous”; e.g., the pH profile of the extent of Mb-H formation remains unexplained. Further- ² We thank the Wellcome Trust (Grant 05731/Z/99/Z) for financial support. * To whom correspondence should be addressed. Phone: (44-1206) 873333 ext 3015. Fax: (44-1206) 872592. E-mail: reedb@essex.ac.uk. ‡ University of Essex. § University of London. 1 Abbreviations: Mb, myoglobin; Mb-H, heme to protein cross- linked myoglobin. 367 Biochemistry 2002, 41, 367-375 10.1021/bi011335b CCC: $22.00 © 2002 American Chemical Society Published on Web 12/08/2001