No abstract

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...