A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide.
Abstract: The reaction between metmyoglobin (metMb) and hydrogen peroxide has been known since the 1950s to produce globin-centered free radicals. The direct electron spin resonance spectrum of a solution of horse metMb and hydrogen peroxide at room temperature consists of a multilined signal that decays in minutes at room temperature. Comparison of the direct ESR spectra obtained from the system under N(2)- and O(2)-saturated conditions demonstrates the presence of a peroxyl radical, identified by its g-value of 2.014. Computer simulations of the spectra recorded 3 s after the mixture of metMb and H(2)O(2) were calculated using hyperfine coupling constants of a(H2,6) = 1.3 G and a(H3,5) = 7.0 G for the ring and a(beta)(H1) = 16.7 G and a(beta)(H2) = 14.2 G for the methylene protons, and are consistent with a highly constrained, conformationally unstable tyrosyl radical. Spectra obtained at later time points contained a mixture of the 3 s signal and another signal that was insufficiently resolved for simulation. Efficient spin trapping with 3, 5-dibromo-4-nitrosobenzenesulfonic acid was observed only when the spin trap was present at the time of H(2)O(2) addition. Spin trapping experiments with either 5,5-dimethyl-1-pyrroline N-oxide (DMPO) or perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)), which have been shown to trap tyrosyl radicals, were nearly equally effective when the spin trap was added before or 10 min after the addition of H(2)O(2). The superhyperfine structure of the ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb confirmed the assignment to a tyrosyl radical. Delayed spin trapping experiments with site-directed mutant myoglobins in which either Tyr-103 or Tyr-146 was replaced by phenylalanine indicated that radical adduct formation with either DMPO or MNP-d(9) requires the presence of Tyr-103 at all time points, implicating that residue as the radical site.
Publication Date: 2000-04-08 PubMed ID: 10754266DOI: 10.1016/s0891-5849(00)00164-7Google Scholar: Lookup
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Summary
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This study explores the reaction between horse metmyoglobin (metMb) and hydrogen peroxide, producing long-lived tyrosyl radicals. They discovered the presence of a peroxyl radical in these reactions and found that efficient spin trapping only occurred when the spin trap was present at the onset of hydrogen peroxide addition.
Overview of the Research
- The research focuses on the reaction between metmyoglobin (metMb) from a horse and hydrogen peroxide. This reaction is seen to produce free radicals in the globin. Free radicals are unstable and highly reactive chemicals that have unpaired electrons.
- The study discovered a peroxyl radical in this reaction, highlighting its presence using nitrogen and oxygen-saturated conditions. This radical was identified by its g-value of 2.014. The g-value is a dimensionless number that provides information about the magnetic characteristics of a radical.
- The paper also presents a simulation of the direct Electron Spin Resonance (ESR) spectra of the solution of metMb and hydrogen peroxide at different time lapses post-mixing. The computer simulations supported the presence of a highly constrained, conformationally unstable tyrosyl radical.
Key Findings
- From the time-lapsed ESR spectra, the researchers found out that efficient spin trapping happened only when the spin trap was present at the onset of hydrogen peroxide addition. Spin trapping is a method used to stabilize free radicals so that they can be analyzed further.
- The researchers tested two different spin traps, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and perdeuterated 2-methyl-2-nitrosopropane (MNP-d(9)). These two have been previously shown to effectively trap tyrosyl radicals. The efficiency of the spin traps was nearly the same whether it was added before or ten minutes after the addition of hydrogen peroxide.
- The study confirms the assignment to a tyrosyl radical through ESR spectra obtained from Pronase-treated MNP-d(9)/*metMb. Pronase is an enzyme used to degrade proteins into their constituent amino acids.
- Experiments with mutated myoglobins, specifically Tyr-103 or Tyr-146 replaced by phenylalanine, shed light on the mechanism of radical adduct formation. The results indicate that Tyr-103 must be present at all times for an effective radical adduct formation with either DMPO or MNP-d(9). This finds points to Tyr-103 as the site of the radical.
Cite This Article
APA
Gunther MR, Sturgeon BE, Mason RP.
(2000).
A long-lived tyrosyl radical from the reaction between horse metmyoglobin and hydrogen peroxide.
Free Radic Biol Med, 28(5), 709-719.
https://doi.org/10.1016/s0891-5849(00)00164-7 Publication
Researcher Affiliations
- Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA. mgunther@hsc.wvu.edu
MeSH Terms
- Animals
- Computer Simulation
- Electron Spin Resonance Spectroscopy / statistics & numerical data
- Free Radicals / metabolism
- Horses
- Hydrogen Peroxide / metabolism
- In Vitro Techniques
- Metmyoglobin / chemistry
- Metmyoglobin / metabolism
- Tyrosine / chemistry
- Tyrosine / metabolism
Citations
This article has been cited 12 times.- Attanzio A, D'Anneo A, Pappalardo F, Bonina FP, Livrea MA, Allegra M, Tesoriere L. Phenolic Composition of Hydrophilic Extract of Manna from Sicilian Fraxinus angustifolia Vahl and its Reducing, Antioxidant and Anti-Inflammatory Activity in Vitro.. Antioxidants (Basel) 2019 Oct 18;8(10).
- Ahmad G, Chami B, El Kazzi M, Wang X, Moreira MTS, Hamilton N, Maw AM, Hambly TW, Witting PK. Catalase-Like Antioxidant Activity is Unaltered in Hypochlorous Acid Oxidized Horse Heart Myoglobin.. Antioxidants (Basel) 2019 Sep 18;8(9).
- Ehrenshaft M, Deterding LJ, Mason RP. Tripping up Trp: Modification of protein tryptophan residues by reactive oxygen species, modes of detection, and biological consequences.. Free Radic Biol Med 2015 Dec;89:220-8.
- Ganini D, Christoff M, Ehrenshaft M, Kadiiska MB, Mason RP, Bechara EJ. Myoglobin-H2O2 catalyzes the oxidation of β-ketoacids to α-dicarbonyls: mechanism and implications in ketosis.. Free Radic Biol Med 2011 Aug 1;51(3):733-43.
- Boutaud O, Moore KP, Reeder BJ, Harry D, Howie AJ, Wang S, Carney CK, Masterson TS, Amin T, Wright DW, Wilson MT, Oates JA, Roberts LJ 2nd. Acetaminophen inhibits hemoprotein-catalyzed lipid peroxidation and attenuates rhabdomyolysis-induced renal failure.. Proc Natl Acad Sci U S A 2010 Feb 9;107(6):2699-704.
- Lardinois OM, Maltby DA, Medzihradszky KF, de Montellano PR, Tomer KB, Mason RP, Deterding LJ. Spin scavenging analysis of myoglobin protein-centered radicals using stable nitroxide radicals: characterization of oxoammonium cation-induced modifications.. Chem Res Toxicol 2009 Jun;22(6):1034-49.
- Suarez J, Ranguelova K, Jarzecki AA, Manzerova J, Krymov V, Zhao X, Yu S, Metlitsky L, Gerfen GJ, Magliozzo RS. An oxyferrous heme/protein-based radical intermediate is catalytically competent in the catalase reaction of Mycobacterium tuberculosis catalase-peroxidase (KatG).. J Biol Chem 2009 Mar 13;284(11):7017-29.
- Ranguelova K, Suarez J, Magliozzo RS, Mason RP. Spin trapping investigation of peroxide- and isoniazid-induced radicals in Mycobacterium tuberculosis catalase-peroxidase.. Biochemistry 2008 Oct 28;47(43):11377-85.
- Connor HD, Sturgeon BE, Mottley C, Sipe HJ Jr, Mason RP. L-tryptophan radical cation electron spin resonance studies: connecting solution-derived hyperfine coupling constants with protein spectral interpretations.. J Am Chem Soc 2008 May 21;130(20):6381-7.
- Lu H, Rusling JF, Hu N. Protecting peroxidase activity of multilayer enzyme-polyion films using outer catalase layers.. J Phys Chem B 2007 Dec 27;111(51):14378-86.
- Bonini MG, Siraki AG, Atanassov BS, Mason RP. Immunolocalization of hypochlorite-induced, catalase-bound free radical formation in mouse hepatocytes.. Free Radic Biol Med 2007 Feb 15;42(4):530-40.
- Svistunenko DA, Cooper CE. A new method of identifying the site of tyrosyl radicals in proteins.. Biophys J 2004 Jul;87(1):582-95.
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