Tyrosyl radical production by myeloperoxidase: a phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins
Introduction
Oxidants produced by phagocytic white blood cells play a critical role in host defenses by destroying invading microbial pathogens and malignant cells (Hurst and Barrette, 1989, Klebanoff, 1980, Babior, 1999). Oxidant generation by phagocytes begins with the generation of superoxide (O2−) by a membrane-associated NADPH oxidase. Superoxide spontaneously or enzymatically dismutates to form hydrogen peroxide (H2O2), a relatively unreactive oxidant.
Phagocytes also secrete the heme protein myeloperoxidase (Kettle and Winterbourn, 1997, Nauseef, 1988), which transforms H2O2 into potent microbicidal oxidants. The enzyme reacts with H2O2 to form compound I, a π-cation radical complex that is reduced by electron donors back to its native oxidation state (Hurst and Barrette, 1989, Kettle and Winterbourn, 1997). In vitro, the myeloperoxidase-H2O2 system can use many electron donors, including halides, reduced pyridine nucleotides, and phenols. As well as protecting the host, this pathway can damage host tissue at sites of inflammation (Heinecke, 1999a). Indeed, many lines of evidence implicate phagocytes in diseases ranging from ischemia-reperfusion injury to cancer to atherosclerosis (Ames et al., 1993, Heinecke, 1999b).
The phenolic amino acid L-tyrosine is present in human blood plasma but was not known to be a substrate for myeloperoxidase. The one-electron oxidation of L-tyrosine by potassium permaganate generates tyrosyl radical (Sealy et al., 1985).When two tyrosyl radicals productively interact, the major product is o,o′-dityrosine (3,3′-dityrosine) (Lehrer and Fasman, 1967), an intensely fluorescent di-amino acid (Anderson, 1966).
This reaction pathway bears remarkable similarities to the chemical generation of phenoxyl radical (Joschek and Miller, 1966, Musso, 1963). In both systems, a one-electron oxidation of a phenol yields a phenoxyl radical, which can form a wide variety of carbon-carbon and carbon-oxygen cross-linked products.
Certain peroxidases oxidize tyrosine to o,o′-dityrosine in vitro (Gross and Sizer, 1959, Deits et al., 1984). The reaction also occurs in vivo, as when the ovoperoxidase of nascent sea urchin embryos generates o,o′-dityrosine cross-links in the protective fertilization envelope by a reaction requiring H2O2 (Foerder and Shapiro, 1977). A cytochrome P450-like enzyme similarly cross-links proteins in the spore wall of the yeast Sacchraromyces (Briza et al., 1986, Briza et al., 1990). It has not been established whether these reactions involve tyrosyl radical and, until recently, it was not known that the myeloperoxidase of activated human phagocytes also produces o,o′-dityrosine cross-links.
Section snippets
Human neutrophils and monocytes use the myeloperoxidase system to generate o,o′-dityrosine
We were intrigued by the possibility that activated phagocytes might use myeloperoxidase to oxidize free L-tyrosine in human extracellular fluid. Therefore, we incubated the enzyme with physiologically plausible concentrations of H2O2 and L-tyrosine. Under these conditions, we readily detected the formation of an intensely fluorescent compound (Heinecke et al., 1993a). We identified it as o,o′-dityrosine by UV-visible spectroscopy, fluorescent spectroscopy, ion exchange chromatography using
Tyrosyl radical generated by myeloperoxidase damages proteins by forming o,o′-dityrosine cross-links
To determine whether o,o′-dityrosine might serve as a marker for oxidative damage to proteins by phagocytes, we investigated the enzyme's ability to form o,o′-dityrosine cross-links in model proteins (Heinecke et al., 1993b). We found that it could cross-link proteins at physiological concentrations of chloride ion and amino acids. Human neutrophils were also able to use the myeloperoxidase-H2O2 system to generate protein-bound dityrosine. As with the synthesis of free o,o′-dityrosine,
Using electron paramagnetic resonance to detect free tyrosyl radical generated by myeloperoxidase
The demonstration that myeloperoxidase uses free L-tyrosine to generate o,o′-dityrosine cross-links indicated that the enzyme converts the amino acid to a freely diffusible, low molecular weight oxidizing intermediate. However, the identity of that intermediate was unclear. Our in vitro studies suggested that it was tyrosyl radical because of the remarkable similarities between phenoxyl radical chemistry and tyrosine oxidation by myeloperoxidase. A second possibility was that the reactions
Human phagocytes use the myeloperoxidase-H2O2 system to generate a family of tyrosyl radical addition products
Tyrosine oxidation products, including o,o′-dityrosine, pulcherosine, isodityrosine, and trityrosine commonly form during post-translational modification of bacterial, yeast, plant, and metazoan proteins (Raven et al., 1971, Pandey and Aronson, 1979, Nomura et al., 1990, Fry, 1982, Foerder and Shapiro, 1977, Briza et al., 1986). In all cases where the biochemical pathway is known, the mechanism involves direct oxidation of protein tyrosyl residues by a heme protein. In contrast, our studies
Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation
Lipid peroxidation has been proposed to play a role in the pathogenesis of disorders ranging from ischemia-reperfusion injury to Alzheimer's disease (Ames et al., 1993). One important example is atherosclerosis, for which an elevated level of low density lipoprotein (LDL), the major carrier of blood cholesterol, is a major risk factor (Brown and Goldstein, 1986). However, many lines of evidence indicate that LDL must be oxidatively modified before it can promote vascular disease (Heinecke, 1998
Proteins in human atherosclerotic tissue are enriched in dityrosine cross-links
Our studies demonstrate that tyrosyl radical generated by myeloperoxidase forms o,o′-dityrosine cross-links in proteins and initiates LDL lipid peroxidation in vitro. Several findings suggest that these processes could also occur in the artery wall. For example, active myeloperoxidase has been detected in human atherosclerotic tissue, where it co-localizes with lipid-laden foam cells, the cellular hallmark of the early atherosclerotic lesion (Daugherty et al., 1994). Immunostaining of
Future directions
Our studies demonstrate that activated human phagocytes use the myeloperoxidase-H2O2 system to oxidize free L-tyrosine to tyrosyl radical. In turn, this oxidizing intermediate acts as a diffusible radical catalyst to initiate lipid peroxidation and create o,o′-dityrosine cross-links in proteins. We used isotope dilution GC/MS to demonstrate that levels of o,o′-dityrosine are elevated in LDL and proteins isolated from human atherosclerotic lesions. These observations suggest that tyrosyl
Acknowledgements
Supported by the National Institutes of Health and the Pharmacia-Monsanto-Searle/Washington University Biomedical Program.
References (53)
NADPH oxidase: an update
Blood
(1999)- et al.
ESR spin-trapping of a protein-derived tyrosyl radical from the reaction of cytochrome c with hydrogen peroxide
J. Biol. Chem.
(1996) - et al.
Oxidative chemistry of peroxynitrite
Methods Enzymol.
(1994) - et al.
NADPH oxidase of neutrophils elevates o,o′-dityrosine cross-links in proteins and urine during inflammation
Arch. Biochem. Biophys.
(2001) - et al.
Dityrosine is a prominent component of the yeast ascospore wall. A proof of its structure
J. Biol. Chem.
(1986) The pecking order of free radicals and antioxidants: lipid peroxidation, alpha-tocopherol, and ascorbate
Arch. Biochem. Biophys.
(1993)- et al.
Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg
J. Biol. Chem.
(1984) - et al.
Mechanism of the formation and proteolytic release of H2O2-induced dityrosine and tyrosine oxidation products in hemoglobin and red blood cells
J. Biol. Chem.
(2001) - et al.
The oxidation of tyramine, tyrosine, and related compounds by peroxidase
J. Biol. Chem.
(1959) Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis
Atherosclerosis
(1998)
Mechanisms of oxidative damage by myeloperoxidase in atherosclerosis and other inflammatory disorders
J. Lab. Clin. Med.
Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages
J. Biol. Chem.
Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: sensitive and quantitative assays for oxidized amino acids in proteins and tissues
Methods Enzymol.
Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation
J. Biol. Chem.
Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway
J. Biol. Chem.
Mass spectrometric quantification of markers for protein oxidation by tyrosyl radical, copper, and hydroxyl radical in low density lipoprotein isolated from human atherosclerotic plaques
J. Biol. Chem.
Caloric restriction attenuates dityrosine cross-linking of cardiac and skeletal muscle proteins in aging mice. Arch. Biochem. Biophys.
Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition
J. Biol. Chem.
Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies
J. Biol. Chem.
Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase
J. Biol. Chem.
Mass spectrometric quantification of 3-nitrotyrosine, orthotyrosine, and o,o′-dityrosine in brain tissue of 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine-treated mice, a model of oxidative stress in Parkinson's disease. J. Biol
Chem.
Occurrence of dityrosine in Tussah silk fibroin and keratin
Biochim. Biophys. Acta
Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein
J. Biol. Chem.
Dityrosine cross-linking promotes formation of stable alpha-synuclein polymers. Implication of nitrative and oxidative stress in the pathogensis of neurodegenerative synucleinopathies. J. Biol
Chem.
The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis
J. Biol. Chem.
Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils
Arch. Biochem. Biophys.
Cited by (106)
An activatable Mn(II) MRI probe for detecting peroxidase activity in vitro and in vivo
2022, Journal of Inorganic BiochemistryRadiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605
2020, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :As in the present study, the experiment was performed in the absence of Cl−, the MPO-Fe (IV) = O (Compound I) is reduced by tyrosine (a molecule that competes for the same site as Cl−) in a single electron reduction to compound II and then another one-electron reduction to native MPO-Fe (III). The rate constant for tyrosine oxidation for the MPO Compound I reaction is comparable to that of its reaction with Cl− [41–43]. The generation of tyrosyl radical in our experiments reflects the generation of Compound I, MPO-Fe (IV) = O, during radiation.
In silico and in vitro antioxidant and cytotoxicity evaluation of oxygenated xanthone derivatives
2020, Arabian Journal of ChemistryBlue autofluorescence in protein aggregates “lighted on” by UV induced oxidation
2019, Biochimica et Biophysica Acta - Proteins and ProteomicsLipoproteins as targets and markers of lipoxidation
2019, Redox Biology