title: Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.
abstract: The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.
authors.affiliation: Medical Clinic, Molecular Cardiology, Medical Center of the Johannes Gutenberg University, Mainz 55131, Germany. andreas.daiber@bioredox.com.
authors.forename: Andreas
authors.initials: A
authors.lastname: Daiber
authors.forename: Steffen
authors.initials: S
authors.lastname: Daub
authors.forename: Markus
authors.initials: M
authors.lastname: Bachschmid
authors.forename: Stefan
authors.initials: S
authors.lastname: Schildknecht
authors.forename: Matthias
authors.initials: M
authors.lastname: Oelze
authors.forename: Sebastian
authors.initials: S
authors.lastname: Steven
authors.forename: Patrick
authors.initials: P
authors.lastname: Schmidt
authors.forename: Alexandra
authors.initials: A
authors.lastname: Megner
authors.forename: Masayuki
authors.initials: M
authors.lastname: Wada
authors.forename: Tadashi
authors.initials: T
authors.lastname: Tanabe
authors.forename: Thomas
authors.initials: T
authors.lastname: Münzel
authors.forename: Serge
authors.initials: S
authors.lastname: Bottari
authors.forename: Volker
authors.initials: V
authors.lastname: Ullrich
mesh_terms: D000818:Animals; D002417:Cattle; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019746:Intramolecular Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D030421:Peroxynitrous Acid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D061987:Sf9 Cells; D018411:Spodoptera; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D013438:Sulfhydryl Compounds; D030421:Peroxynitrous Acid; C002744:3-nitrotyrosine; D014443:Tyrosine; D003577:Cytochrome P-450 Enzyme System; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase