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A free radical mechanism for s-nitrosohemoglobin formation from nitrite

Grant number: 12/20485-6
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): December 01, 2012
Effective date (End): April 30, 2014
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:José Carlos Toledo Junior
Grantee:Ana Carolina Fonseca
Home Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:10/00550-2 - Mechanisms of cellular s-nitrosation from free nitric oxide. Involviment of transition metals and reactive species, AP.JP

Abstract

Recent studies show that the reaction of deoxyhemoglobin and nitrite yields small but sustained amounts of s-nitrosohemoglobin (bCys93-(NO)HbFe2+), a Hb species where the Hb b chain cystein 47 residue is found s-nitrosated. It has been hypothesized that s-nitrosohemoglobin somehow releases NO or NO equivalents that promotes vasodilation during the hemoglobin R/T allosteric transition upon deoxygenation. Thus, hemoglobin would exert the double function of delivering O2 and the potent vasodilator NO, simultaneously facilitating its own path through the capillaries and promoting tissue oxygenation. Several knowledge gaps need to be filled before this hypothesis can be fully accepted though. This project focuses on the formation of s-nitrosohemoglobin. The reaction of Hb with nitrite is well studied and occurs through reaction 1-3. This route necessarily produces free NO (reaction 3) and this is probably the predominant pathway, although, NO is rapidly trapped by excess deoxyhemoglobin, a very rapid reaction that produces the very stable nitrosyl hemoglobin species (bCys93HbFe2+-NO). bCys93 HbFe2+ + NO2 6’bCys93HbFe2+-NO2 6(1)bCys93HbFe2+-NO2 6 + 2H+’ bCys93 HbFe3+-NO + H2O(2)bCys93HbFe3+-NO’bCys93HbFe3+ + NO(3)bCys93 HbFe3+-NO + NO2- ’ bCys93HbFe2+-NO + NO2 (4)bCys93S-HbFe2+ + NO2 ’ bCys93S*-HbFe2+ + NO2-(5)bCys93S*-HbFe2+ + NO ’ bCys93SNO-HbFe2+ (6)Interestingly, Ford et al found that the intermediate bCys93HbFe3+-NO can be intercepted and reduced by nitrite (reaction 4) yielding nitrosyl hemoglobin and, importantly, the free radical nitrogen dioxide (NO2). The formation of this radical prompted us to hypothesize that NO2 may react with b chain cystein 47 residue thiol group to produce the thyil radical species bCys93S*-HbFe2+ (reaction 5) that, in turn, may recombine with NO (from reaction 3) to form s-nitrosohemoglobin (reaction 6). Thus, we suggest a radical mechanism for formation of s-nitrosohemoglobin which is supported by well known reactions between Hb and nitrite. This hypothesis is consistent with the low amounts of s-nitrosohemoglobin formed since it is actually a competitive route and depends on the predominant release of NO pathway. The investigation of this mechanism is relevant and may fill one of the main gaps on the so-called Hb double functionality hypothesis.