A free radical mechanism for s-nitrosohemoglobin formation from nitrite

• Grant number: 12/20485-6
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: December 01, 2012
• End date: April 30, 2014
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: José Carlos Toledo Junior
• Grantee: Ana Carolina Fonseca
• Host 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 cysteine 47 residue is found s-nitrosated. It has been hypothesized that s-nitrosohemoglobin somehow releases NO or NO equivalents that promote 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 reactions 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 cysteine 47 residue thiol group to produce their 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 the formation of s-nitroso hemoglobin 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 in the so-called Hb double functionality hypothesis. (AU)