The oxyhemoglobin reaction of nitric oxide

Abstract

The oxidation of nitric oxide (NO) to nitrate by oxyhemoglobin is a fundamental reaction that shapes our understanding of NO biology. This reaction is considered to be the major pathway for NO elimination from the body; it is the basis for a prevalent NO assay; it is a critical feature in the modeling of NO diffusion in the circulatory system; and it informs a variety of therapeutic applications, including NOinhalation therapy and blood substitute design. Here we show that, under physiological conditions, this reaction is of little significance. Instead, NO preferentially binds to the minor population of the hemoglobin's vacant hemes in a cooperative manner, nitrosylates hemoglobin thiols, or reacts with liberated superoxide in solution. In the red blood cell, superoxide dismutase eliminates superoxide, increasing the yield of Snitrosohemoglobin and nitrosylated hemes. Hemoglobin thus serves to regulate the chemistry of NO and maintain it in a bioactive state. These results represent a reversal of the conventional view of hemoglobin in NO biology and motivate a reconsideration of fundamental issues in NO biochemistry and therapy.The chemistry of nitric oxide (NO) interactions with Hb has served as a ubiquitous model within the field of NO biochemistry. For example, the oxidative interaction of NO with oxyhemoglobin (oxyHb) to produce nitrate is considered to be the major route of NO catabolism (1-3) as well as a reliable method for assaying NO (4); likewise the unique ability of NO to induce displacement of a trans-imidazole heme ligand has been proposed as key to its activation of guanylyl cyclase (5). In the specific realm of the cardiovascular system, these reactions: are fundamental elements of models for NO diffusion (6, 7); played a crucial role in the identification of endothelium derived relaxing factor (6-9); and inform a variety of therapeutic applications, including NO-inhalation therapy (10, 11) and blood substitute design (12,13).Measurements of the rates of these reactions show that the NO-mediated oxidation of oxyHb to methemoglobin (metHb) is kinetically competitive with the binding of NO to unoccupied hemes in Hb-with specific rate constants of 3.7 ϫ 10 ) (16). Such a rapid route of NO metabolism is, however, difficult to reconcile with mammalian NO production rates (17), which are orders of magnitude too low to sustain physiological NO levels (10 nM-1 M) (7,(18)(19)(20), were NO to be freely consumed in these reactions. Previous studies of the NO oxyHb reaction, however, had been performed with NO concentrations 10-fold greater than protein (16). Under physiological conditions, the concentration ratio is starkly different, with NO concentrations 1,000-fold lower than Hb (20). Moreover, there is always a population of heme sites that are unoccupied. In highly oxygenated Hb, as found in arterial blood, this population is small (Ϸ1%) but is nevertheless in excess of NO. The influence of these vacant hemes, in the physiological situation, cannot be ignored; they might successfully compete f...

Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

“…The midgut conditions resemble that of a cell-free Hb system since the RBCs nearest to the midgut epithelium, the source of · NO, are actively digested and lysed. The higher concentrations of Hb in the midgut together with high local concentrations of · NO produced by the midgut epithelium, could support nitrosylHb formation as described by Gow et al [34]. Further, the reaction of · NO with oxyHb to form nitrosylHb may be favored in the parasiteinfected midgut where in addition to high local · NO fluxes, Hb is freed from RBCs and sequestered by disulfide bond-dependent aggregation during digestion.…”

“…The addition of superoxide dismutase (SOD) to Hb increases the yield of total · NO bound to Hb [34]. SOD activity is increased by bloodfeeding in A. gambiae by 0.95-1.94-fold at 12h pBM and 2.1-2.5-fold at 24h pBM relative to unfed controls [35], suggesting that mosquito SOD could facilitate · NO binding to Hb.…”

The role of β93 Cys in the inhibition of Hb S fiber formation

“…This model stems from the observation that SNO-Hb formation is favored when Hb is in the R-or fully liganded state, while HbNO formation is favored when Hb is in the T-state [17]. In this model, NO transport is related to oxygen tension, which implies that a detectable concentration of NO exists in the blood, and that NO activity is preserved by binding to Hb [19]. The inhibitory effect in SCD is attributed to the formation of SNO-Hb, which stabilizes the Rstate and increases HbS fiber solubility.…”

Interactions of nitrosylhemoglobin and carboxyhemoglobin with erythrocyte

“…It is therefore necessary to preserve NO bioactivity in the proximity of a pool of hemoglobin (∼10 mM) in the lumen. Several solutions to this paradox have been proposed, including i) the diffusion and transport limitation arises from the encapsulation of hemoglobin (Hb) in erythrocyte [8,9,10,11,12,13,14,5,15,16,17,18], ii) the formation of S-nitroso-hemoglobin (SNO-Hb) from the intramolecular transfer of nitrosylhemoglobin (HbFe II NO) [19,20,21,22,23,24,5,25], and iii) the NO bioactivity transduced from nitrite [26,27,28,29]. In the latter two cases, the formation of HbFe II NO can be argued to play a role for preserving NO bioactivity.…”