Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?
Résumé
Human neuroglobin (NGB), a recently discovered heme protein of the globin family containing a six-coordinated heme, is expressed in the nervous tissue with a yet unclear physiological function. Besides being involved in neuronal oxygen homeostasis, NGB is thought to act as a scavenger of reactive species. Herein we report on the reactivity of metNGB, which can accumulate in vivo by reaction of oxyNGB with nitric oxide, towards nitrite and H 2}O 2}. Nitrite coordination to the heme accounts for the activity of metNGB in the nitration of phenolic substrates. The two metNGB forms, with and without the internal disulfide bond between Cys(46)CD7 and Cys(55)D5, exhibit different reactivity, the former being more efficient in activating NO 2} -}. The kinetics of the reactions, the nitrite-binding studies, and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate, at pathophysiological concentration of nitrite and H 2}O 2}, an active species with the chemical properties of peroxynitrite. Without external substrates, the targets of the reactive species generated by the metNGB/NO 2} -}/H 2}O 2} system are endogenous tyrosine (transformed into 3-nitrotyrosine) and cysteine (oxidized to sulfinic and sulfonic acids) residues. The endogenous modifications were characterized by HPLC-MS/MS analysis of metNGB reacted with nitrite and H 2}O 2} in various conditions. The internal S-S bond affects the functional properties of the protein. Therefore, metNGB acts not only as scavenger of toxic species, but also as target of the self-generated reactive species: the protein self-modification may be related or impair its postulated neuroprotective activity.
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