Elsevier

Toxicology

Volume 177, Issue 1, 1 August 2002, Pages 11-22
Toxicology

Tyrosyl radical production by myeloperoxidase: a phagocyte pathway for lipid peroxidation and dityrosine cross-linking of proteins

https://doi.org/10.1016/S0300-483X(02)00192-0Get rights and content

Abstract

To kill invading bacteria, viruses, and fungi, phagocytes secrete hydrogen peroxide (H2O2) and the heme enzyme myeloperoxidase. We have explored the possibility that myeloperoxidase might use H2O2 to convert L-tyrosine to tyrosyl radical. Activated human neutrophils and monocytes used the system to oxidize free L-tyrosine to o,o′-dityrosine, a stable product of tyrosyl radical. Protein-bound tyrosyl residues exposed to myeloperoxidase, H2O2, and l-tyrosine were also oxidized to o,o′-dityrosine. The cross-linking reaction required free L-tyrosine, suggesting that myeloperoxidase converts the amino acid to a diffusible radical catalyst that promotes protein oxidation. We used electron paramagnetic resonance to provide direct evidence that the oxidizing intermediate is free tyrosyl radical. Myeloperoxidase-generated tyrosyl radical also initiates lipid peroxidation, suggesting that activated phagocytes might also be able to oxidize lipids in host tissues. Moreover, myeloperoxidase is present and active in human atherosclerotic tissue, and levels of protein-bound dityrosine are elevated in such lesions. Our recent studies indicate that activated neutrophils use oxidants generated by the phagocyte NADPH oxidase to produce protein-bound dityrosine during acute inflammation. Collectively, these findings suggest that generation of tyrosyl radical by myeloperoxidase allows activated phagocytes to damage both proteins and lipids. Elevated levels of o,o′-dityrosine have been detected in inflammatory lung disease, neurodegenerative disorders, and aging. Thus, oxidation of tyrosine to tyrosyl radical might play a role in the pathogenesis of many diseases.

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 (O2radical dot) by a membrane-associated NADPH oxidase. Superoxide spontaneously or enzymatically dismutates to form hydrogen peroxide (H2O2), a relatively unreactive oxidant.NADPH+O2→NADP++O2→H2O2

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)

  • J.W. Heinecke

    Mechanisms of oxidative damage by myeloperoxidase in atherosclerosis and other inflammatory disorders

    J. Lab. Clin. Med.

    (1999)
  • J.W. Heinecke et al.

    Dityrosine, a specific marker of oxidation, is synthesized by the myeloperoxidase-hydrogen peroxide system of human neutrophils and macrophages

    J. Biol. Chem.

    (1993)
  • J.W. Heinecke et al.

    Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: sensitive and quantitative assays for oxidized amino acids in proteins and tissues

    Methods Enzymol.

    (1999)
  • T.G. Huggins et al.

    Formation of o-tyrosine and dityrosine in proteins during radiolytic and metal-catalyzed oxidation

    J. Biol. Chem.

    (1993)
  • J.S. Jacob et al.

    Human phagocytes employ the myeloperoxidase-hydrogen peroxide system to synthesize dityrosine, trityrosine, pulcherosine, and isodityrosine by a tyrosyl radical-dependent pathway

    J. Biol. Chem.

    (1996)
  • C. Leeuwenburgh et al.

    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.

    (1997)
  • C. Leeuwenburgh et al.

    Caloric restriction attenuates dityrosine cross-linking of cardiac and skeletal muscle proteins in aging mice. Arch. Biochem. Biophys.

    (1997)
  • L.J. Marnett et al.

    Arachidonic acid oxygenation by COX-1 and COX-2. Mechanisms of catalysis and inhibition

    J. Biol. Chem.

    (1999)
  • L.A. Marquez et al.

    Kinetics of oxidation of tyrosine and dityrosine by myeloperoxidase compounds I and II. Implications for lipoprotein peroxidation studies

    J. Biol. Chem.

    (1995)
  • M.L. McCormick et al.

    Electron paramagnetic resonance detection of free tyrosyl radical generated by myeloperoxidase, lactoperoxidase, and horseradish peroxidase

    J. Biol. Chem.

    (1998)
  • S. Pennathur et al.

    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.

    (1999)
  • D.J. Raven et al.

    Occurrence of dityrosine in Tussah silk fibroin and keratin

    Biochim. Biophys. Acta

    (1971)
  • M.L. Savenkova et al.

    Tyrosyl radical generated by myeloperoxidase is a physiological catalyst for the initiation of lipid peroxidation in low density lipoprotein

    J. Biol. Chem.

    (1994)
  • J.M. Souza et al.

    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.

    (2000)
  • B.E. Sturgeon et al.

    The fate of the oxidizing tyrosyl radical in the presence of glutathione and ascorbate. Implications for the radical sink hypothesis

    J. Biol. Chem.

    (1998)
  • C.C. Winterbourn et al.

    Myeloperoxidase-dependent generation of a tyrosine peroxide by neutrophils

    Arch. Biochem. Biophys.

    (1997)
  • Cited by (106)

    • Radiation activates myeloperoxidase (MPO) to generate active chlorine species (ACS) via a dephosphorylation mechanism - inhibitory effect of LGM2605

      2020, Biochimica et Biophysica Acta - General Subjects
      Citation 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.

    • Blue autofluorescence in protein aggregates “lighted on” by UV induced oxidation

      2019, Biochimica et Biophysica Acta - Proteins and Proteomics
    View all citing articles on Scopus
    View full text