Article pubs.acs.org/biochemistry
Red Blood Cell Membrane-Facilitated Release of Nitrite-Derived Nitric Oxide Bioactivity Maria T. Salgado, Zeling Cao, Enika Nagababu, Joy G. Mohanty, and Joseph M. Rifkind* Molecular Dynamics Section, National Institute on Aging, National Institutes of Health, 251 Bayview Boulevard, Baltimore, Maryland 21224, United States ABSTRACT: The reduction of nitrite by deoxyhemoglobin to nitric oxide (NO) has been proposed as a mechanism for the transfer of NO bioactivity from the red blood cell (RBC) to the vasculature. This transfer can increase vascular dilatation. The major challenge to this hypothesis is the very efficient scavenging of NO by hemoglobin, which prevents the release of NO from RBCs. Previous studies indicate that the reaction of nitrite with deoxyhemoglobin produces two metastable intermediates involving nitrite bound to deoxyhemoglobin and a hybrid intermediate [Hb(II)NO+ ↔ Hb(III)NO] where the nitrite is reduced, but unavailable to react with hemoglobin. We have now shown how unique properties of these intermediates provide a pathway for the release of NO bioactivity from RBCs. The high membrane affinity of these intermediates (>100-fold greater than that of deoxyhemoglobin) places these intermediates on the membrane. Furthermore, membrane-induced conformational changes of the nitrite-reacted intermediates facilitate the release of NO from the hybrid intermediate and nitrite from the nitrite-bound intermediate. Increased membrane affinity, coupled with facilitated dissociation of NO and nitrite from the membrane-bound intermediates, provides the first realistic mechanism for the potential release of NO and nitrite from the RBC and their potential transfer to the vasculature.
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require that NO bioactivity be released from the RBC. These hypotheses, therefore, must be able to avoid the very efficient scavenging of NO by both oxyhemoglobin (oxyHb) and deoxyhemoglobin (deoxyHb).12−14 The Stamler hypothesis bypassed this difficulty by S-nitrosation of the β-93 thiol group15 and the subsequent transfer of this NO to membrane and plasma thiol groups. Several studies have also proposed mechanisms for nitrite-associated release of NO bioactivity from the RBC without actually releasing NO. It was, thus, suggested that the reaction of nitrite with deoxyHb could result in the release of N2O3.16 Nitrite can also result in the release of ATP.17 No mechanism has, however, been proposed that satisfactorily explains the release of NO from RBCs. In this paper, a potential mechanism for the actual release of NO from the RBC to the vasculature is provided for the first time. We previously demonstrated that during the reaction of nitrite with hemoglobin (Hb) only ∼25% of the nitrite is converted to NO, which immediately reacts with deoxyHb producing Hb(II)NO. The rest of the nitrite-reacted Hb forms two metastable intermediates that can, under the proper conditions, release NO or nitrite, retaining potential NO bioactivity in a form that is not quenched by Hb.10 The first intermediate involving
itric oxide (NO) as a vasodilator plays a major role in regulating blood flow and vascular tone.1 The primary route for the synthesis of NO in the circulatory system is endothelial nitric oxide synthase (eNOS).2 NO synthesized by eNOS can diffuse to the smooth muscle cells or into the plasma. Diffusion of NO into the smooth muscle cells results in vasodilatation. The NO that diffuses into the plasma has a halflife in whole blood of
