Oxidized lipoproteins may play an important role in the pathogenesis of atherosclerosis. Elevated levels of 3-chlorotyrosine, a specific end product of the reaction between hypochlorous acid (HOCl) and tyrosine residues of proteins, have been detected in atherosclerotic tissue. Thus, HOCl generated by the phagocyte enzyme myeloperoxidase represents one pathway for protein oxidation in humans. One important target of the myeloperoxidase pathway may be high density lipoprotein (HDL), which mobilizes cholesterol from artery wall cells. To determine whether activated phagocytes preferentially chlorinate specific sites in HDL, we used tandem mass spectrometry (MS/MS) to analyze apolipoprotein A-I that had been oxidized by HOCl. The major site of chlorination was a single tyrosine residue located in one of the protein's YXXK motifs (where X represents a nonreactive amino acid). To investigate the mechanism of chlorination, we exposed synthetic peptides to HOCl. The peptides encompassed the amino acid sequences YKXXY, YXXKY, or YXXXY. MS/MS analysis demonstrated that chlorination of tyrosine in the peptides that contained lysine was regioselective and occurred in high yield if the substrate was KXXY or YXXK. NMR and MS analyses revealed that the N(epsilon) amino group of lysine was initially chlorinated, which suggests that chloramine formation is the first step in tyrosine chlorination. Molecular modeling of the YXXK motif in apolipoprotein A-I demonstrated that these tyrosine and lysine residues are adjacent on the same face of an amphipathic alpha-helix. Our observations suggest that HOCl selectively targets tyrosine residues that are suitably juxtaposed to primary amino groups in proteins. This mechanism might enable phagocytes to efficiently damage proteins when they destroy microbial proteins during infection or damage host tissue during inflammation.

Matrix metalloproteinases (MMPs) function in homeostatic and repair processes, but unregulated catalysis by these extracellular proteinases leads to the pathological destruction of tissue proteins. An important mechanism for controlling enzyme activity might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase system of phagocytes. We have shown that inactivation of MMP-7 (matrilysin) by HOCl coincides with the formation of a novel oxidation product, WG؊4, through modification of adjacent tryptophan and glycine residues and loss of 4 atomic mass units. Here, we use mass spectrometry, UV/visible spectroscopy, hydrogen-deuterium exchange, and NMR spectroscopy to investigate the formation and structure of WG؊4. For the initial step, HOCl chlorinates the indole ring of tryptophan. The resulting 3-chloroindolenine generates a previously unknown cyclic indole-amide species, in which tryptophan cross-links to the main chain nitrogen of the adjacent glycine residue to form an aromatic six-membered ring. WG؊4 kinks and stiffens the peptide backbone, which may hinder the interaction of substrate with the catalytic pocket of MMP-7. Our observations indicate that specific structural motifs are important for controlling protein modification by oxidants and suggest that pericellular oxidant production by phagocytes might limit MMP activity during inflammation. Matrix metalloproteinases (MMPs)1 play a central role in the proteolytic regulation of proteins involved in inflammation and repair, in the turnover of extracellular matrix, and in pathological destruction of tissue proteins (1). Dysregulation of MMPs is implicated in the pathogenesis of destructive diseases, such as aneurysms, emphysema, and arthritis, as well as tumor growth and metastasis (2-4).MMPs are synthesized as zymogens (1). The prodomain of latent MMPs interacts with the zinc ion in the catalytic domain. This interaction is critical to maintaining the enzyme in an inactive state. The mechanisms that control the activation and subsequent inactivation of MMPs have generally been attributed to other proteinases and protein inhibitors, respectively (1). However, the physiological mechanisms regulating MMP proteolysis remain largely unknown. Indeed, reactive oxygen and nitrogen intermediates regulate MMP activity in vitro (5-9) raising the possibility that similar reactions control MMP activity in vivo.We previously demonstrated that hypochlorous acid (HOCl), a potent oxidant generated by neutrophils, monocytes, and macrophages (10 -12), can activate pro-MMP-7 (promatrilysin) by converting the thiol residue of the prodomain to the corresponding sulfinic acid (7). Recent studies suggest that oxygenation of pro-MMPs may be physiologically relevant (13).The modification of MMPs provides a relevant paradigm illustrating how HOCl can regulate protein function by targeting specific amino acids within critical domains. Although HOCl can initially activate pro-MMPs, subsequent inhibition of enzyme activity is the predominant effect at physiol...