Recent evidence suggests that the reaction between nitrite and deoxygenated hemoglobin provides a mechanism by which nitric oxide is synthesized in vivo. This reaction has been previously defined to follow second order kinetics, although variable product stoichiometry has been reported. In this study we have re-examined this reaction and found that under fully deoxygenated conditions the product stoichiometry is 1:1 (methemoglobin:nitrosylhemoglobin), and unexpectedly, the kinetics deviate substantially from a simple second order reaction and exhibit a sigmoidal profile. The kinetics of this reaction are consistent with an increase in reaction rate elicited by heme oxidation and iron-nitrosylation. In addition, conditions that favor the "R" conformation show an increased rate over conditions that favor the "T" conformation. The reactivity of nitrite with heme is clearly more complex than has been previously realized and is dependent upon the conformational state of the hemoglobin tetramer, suggesting that the nitrite reductase activity of hemoglobin is under allosteric control.The reaction between nitrite and deoxyhemoglobin (deoxyHb 1 NO] 2ϩ ) (1) through the intermediacy of nitric oxide (NO) (2). This reaction has recently been invoked to explain nitrite-mediated effects on blood flow at rest and during exercise. At supra-and near-physiologic concentrations, nitrite produces increased blood flow in a dose-dependent fashion, regardless of NO synthase inhibition or metabolic demand (3). Furthermore, HbNO production, formed from nitrite infusion, was shown to be inversely proportional to hemoglobin oxygen saturation (3). An important characteristic of nitrite is that HbNO formation only occurs with deoxyHb and not oxygenated hemoglobin (oxyHb or Hb[Fe II O 2 ] 2ϩ ), suggesting an oxygen-dependence to the NOforming reaction. The reaction of nitrite with oxyHb occurs via a complex autocatalytic mechanism that has not yet been fully elucidated (4). The potential nitrite-reductase activity of deoxyHb has previously been invoked as a potential source of bioactive NO (5). The reaction between nitrite and deoxyHb was extensively examined by Doyle et al. (2). These investigators concluded that the reaction was second order and that the major products of the reaction were metHb and HbNO. Their observations were largely consistent with the reactions shown in Equations 1-4. As reaction 3 is extremely fast (k 3 ϳ 4 ϫ 10 7 ), the kinetics of the reaction are dictated by the second order reaction shown in Equation 2, which was calculated to have a rate constant of 1. In this study we have re-examined this reaction under conditions of molar excess Hb to nitrite and vice versa. In contrast to the earlier study (2), we concluded that although the initial rate of the reaction has a first order dependence on both nitrite and hemoglobin concentrations, the kinetics profile does not fit a second order/reaction but exhibits autocatalytic kinetics. In addition we found that under strictly anaerobic conditions the stoichiometry o...

Nitric oxide has been proposed to be transported by hemoglobin as a third respiratory gas and to elicit vasodilation by an oxygenlinked (allosteric) mechanism. For hemoglobin to transport nitric oxide bioactivity it must capture nitric oxide as iron nitrosyl hemoglobin rather than destroy it by dioxygenation. Once bound to the heme iron, nitric oxide has been reported to migrate reversibly from the heme group of hemoglobin to the ␤-93 cysteinyl residue, in response to an oxygen saturation-dependent conformational change, to form an S-nitrosothiol. However, such a transfer requires redox chemistry with oxidation of the nitric oxide or ␤-93 cysteinyl residue. In this article, we examine the ability of nitric oxide to undergo this intramolecular transfer by cycling human hemoglobin between oxygenated and deoxygenated states. Under various conditions, we found no evidence for intramolecular transfer of nitric oxide from either cysteine to heme or heme to cysteine. In addition, we observed that contaminating nitrite can lead to formation of iron nitrosyl hemoglobin in deoxygenated hemoglobin preparations and a radical in oxygenated hemoglobin preparations. Using 15 N-labeled nitrite, we clearly demonstrate that nitrite chemistry could explain previously reported results that suggested apparent nitric oxide cycling from heme to thiol. Consistent with our results from these experiments conducted in vitro, we found no arterial͞venous gradient of iron nitrosyl hemoglobin detectable by electron paramagnetic resonance spectroscopy. Our results do not support a role for allosterically controlled intramolecular transfer of nitric oxide in hemoglobin as a function of oxygen saturation. How is the activity of the endothelium-derived relaxation factor, nitric oxide (NO), preserved if an abundant scavenger, hemoglobin (Hb), reacts so quickly with it? This question has puzzled many investigators ever since the identification of NO as the endothelium-derived relaxation factor was made (1-4). NO reacts with Hb via the following reactions:where Fe(II) and Fe(III) refer to the ferrous and ferric forms of the iron in the heme group of Hb, respectively. The rate constants for these reactions are 2.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (5) and 6-9 ϫ 10 7 M Ϫ1 ⅐s Ϫ1 at 20°C (6, 7) for Eqs. 1 and 2, respectively. The second reaction destroys NO activity through the formation of nitrate. The activity of NO is potentially conserved by the reaction described by Eq. 1, but to function it must come off the heme and get out of the RBC (where the Hb concentration is Ϸ0.02 M in heme) before undergoing the fast reactions described by Eqs. 1 and 2. Thus, scavenging of NO in blood can be predicted to be a significant NO sink and poses the problem of how NO can function as the endothelium-derived relaxation factor (8).One possible resolution of this paradox would be that NO produced in endothelial cells acts only locally and is thus immune to scavenging by Hb, but a variety of evidence indicating increased vasoconstriction when free Hb or Hb substitutes are in...

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