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...

Previous studies of the interactions of NO with human hemoglobin have implied the predominance of reaction channels that alternatively eliminate NO by converting it to nitrate, or tightly complex it on the ␣ subunit ferrous hemes. Both channels could effectively quench NO bioactivity. More recent work has raised the idea that NO groups can efficiently transfer from the hemes to cysteine thiols within the ␤ subunit (cys␤-93) to form bioactive nitrosothiols. The regulation of NO function, through its chemical position in the hemoglobin, is supported by response to oxygen and to redox agents that modulate the molecular and electronic structure of the protein. In this article, we focus on reactions in which Fe(III) hemes could provide the oxidative requirements of this NO-group transfer chemistry. We report a detailed investigation of the reductive nitrosylation of human met-Hb, in which we demonstrate the production of S-nitroso (SNO)-Hb through a heme-Fe(III)NO intermediate. The production of SNO-Hb is strongly favored (over nitrite) when NO is gradually introduced in limited total quantities; in this situation, moreover, heme nitrosylation occurs primarily within the ␤ subunits of the hemoglobin tetramer. SNO-Hb can similarly be produced when Fe(II)NO hemes are subjected to mild oxidation. The reaction of deoxygenated hemoglobin with limited quantities of nitrite leads to the production of ␤ subunit Fe(II)NO hemes, with SNO-Hb produced on subsequent oxygenation. The common theme of these reactions is the effective coupling of heme-iron and NO redox chemistries. Collectively, they establish a connectivity between hemes and thiols in Hb, through which NO is readily dislodged from storage on the heme to form bioactive SNO-Hb.T he transfer of NO groups within human hemoglobin from hemes to cys(␤-93) thiols to form a bioactive nitrosothiol represents a novel intramolecular biochemistry that is both of fundamental interest and has considerable implications for understanding the physiological effects of NO in the regulation of vascular tension and blood f low. A requirement of this transfer, common to biological S-nitrosylation (1), is the redox activation of the NO group (2). In this article, we report the results of experiments that probe the idea that heme-iron valence change can support the oxidative requirements of NO-group transfer and thus efficiently lead to the production of S-nitroso (SNO)-Hb. As a model of the reaction between ferric hemes and NO, the reductive nitrosylation of human methemoglobin is examined in detail. Product distribution assays reveal that SNO-Hb is formed as a nitrosation product, which, moreover, is substantially favored over NO 2 Ϫ when NO is gradually introduced as a limiting reagent; furthermore, in this situation, heme nitrosylation occurs primarily within the ␤ subunits of the Hb tetramer. A kinetic analysis unambiguously reveals the intermediacy of heme-Fe(III)NO in this reaction. To extend our observations to reactions that could mimic this chemistry but do not require an accumula...