Hemoglobin conformation couples erythrocyte S -nitrosothiol content to O ₂ gradients

Abstract

It is proposed that the bond between nitric oxide (NO) and the Hb thiol Cys-␤ 93 (SNOHb) is favored when hemoglobin (Hb) is in the relaxed (R, oxygenated) conformation, and that deoxygenation to tense (T) state destabilizes the SNOHb bond, allowing transfer of NO from Hb to form other (vasoactive) S-nitrosothiols (SNOs). However, it has not previously been possible to measure SNOHb without extensive Hb preparation, altering its allostery and SNO distribution. Here, we have validated an assay for SNOHb that uses carbon monoxide (CO) and cuprous chloride (CuCl)-saturated Cys. This assay is specific for SNOs and sensitive to 2-5 pmol. Uniquely, it measures the total SNO content of unmodified erythrocytes (RBCs) (SNORBC), preserving Hb allostery. In room air, the ratio of SNORBC to Hb in intact RBCs is stable over time, but there is a logarithmic loss of SNORBC with oxyHb desaturation (slope, 0.043). This decay is accelerated by extraerythrocytic thiol (slope, 0.089; P < 0.001). SNORBC stability is uncoupled from O2 tension when Hb is locked in the R state by CO pretreatment. Also, SNORBC is increased Ϸ20-fold in human septic shock (P ‫؍‬ 0.002) and the O2-dependent vasoactivity of RBCs is affected profoundly by SNO content in a murine lung bioassay. These data demonstrate that SNO content and O2 saturation are tightly coupled in intact RBCs and that this coupling is likely to be of pathophysiological significance.sepsis ͉ nitric oxide ͉ vascular physiology E vidence has accumulated for an S-nitrosothiol (SNO)-based vascular signaling system in which hemoglobin (Hb) reactions with nitric oxide (NO) transduce redox gradients into bioactivities (1-6). There is agreement that human Hb undergoes Snitrosylation at Cys-␤ 93 (3,(7)(8)(9)(10)(11). Erythrocytes are proposed to couple O 2 tension to the distribution of NO activities (such as control of blood flow) by linking the allosteric transition of Hb (12, 13) to conformation-dependent changes in the redox activity of this Cys-␤ 93 (13-18) and the stereochemistry of this SNO bond at Cys-␤ 93 (6, 7). Indeed, Cys-␤ 93 SNO in human Hb (SNOHb) can be crystallized only with the Hb tetramer in the relaxed (R, oxygenated) conformation; the SNO bond is unstable with Hb in the tense (T, deoxygenated) conformation (7). These observations support a paradigm in which NO binding to Cys-␤ 93 is favored in the R state and NO binding to Fe(II) (and͞or transnitrosation to an alternate thiol) is favored in the T state (19-21). Thus, the change in stability of Cys-␤ 93 SNO during Hb transition between R and T states may serve to couple regional O 2 gradients to the deployment or quenching of NO bioactivities in the microcirculation (2, 6, 22).However, assaying SNOHb has been problematic. First, detection of the SNO bond has required dilution and͞or pretreatment of Hb to (i) control for artifactual identification of nitrite and Fenitrosyl species and (ii) prevent autocapture of NO on Fe during analysis (8,19,(23)(24)(25). As a result, attempts to quantify Cys-␤ 93 SNO density can be biased ...

A nitric oxide processing defect of red blood cells created by hypoxia: Deficiency of S-nitrosohemoglobin in pulmonary hypertension

“…Because it is Hb O 2 saturation, not the partial pressure of O 2 (pO 2 ), that is coupled to blood flow in vivo (1,3) it has been deduced that RBCs may serve as O 2 sensors within the integrated vascular system. In support of this idea, it has been shown recently that RBCs can act as O 2 -responsive transducers of vasodilator and vasoconstrictor activity (4)(5)(6)(7)(8)(9)(10), at least partly by modulating the availability of NO (6-8, 10, 11). According to these studies, RBCs release NO bioactivity under hypoxia and sequester it at hyperoxia.…”

“…Transnitrosylation of AE1 by SNO-Hb involves a direct protein-protein interaction. The steps by which the NO group is subsequently transferred to the vessel wall are not yet established, although recent studies suggest that membrane SNO becomes accessible to plasma reactants, including glutathione (10,20).Hb is continuously cycling in vivo between oxygenated and deoxygenated states that influence the propensity for binding vs. release of NO groups (5,16,18,21). In support of this proposition, SNO-Hb levels have been found by several independent groups and methods (3) to be higher in oxygenated blood than in deoxygenated blood of adults (6, 8, 10) and newborns (22) and to vary as a function of tissue oxygen saturations (6,8,10,23).…”

An S -nitrosothiol (SNO) synthase function of hemoglobin that utilizes nitrite as a substrate

“…Vasodilation (of aortic or pulmonary artery rings) by RBCs in bioassay is rapid, in keeping with the temporal requirements of arterial-venous transit (in seconds) (3,4). Moreover, when infused into animals, RBCs increase blood flow and improve oxygenation, an indication that RBCs elicit vasodilation in both the systemic and pulmonary circulations (1,(4)(5)(6). Collectively, the observation of vasoconstriction by RBCs at higher pO 2 but graded vasodilation with increasing hypoxia appears to provide a mechanism for matching blood flow to metabolic demand in the peripheral tissues and for matching ventilation to perfusion in the lungs (7).…”

“…This exchange of NO groups occurs primarily in the T state (deoxyHb; high spin) of Hb (3,6,18), which exposes the ␤93cysNO to solvent and adjusts heme͞SNO redox coupling (7,18). As a result, NO group transfer is graded with the degree of hypoxia (6,19).…”

“…This exchange of NO groups occurs primarily in the T state (deoxyHb; high spin) of Hb (3,6,18), which exposes the ␤93cysNO to solvent and adjusts heme͞SNO redox coupling (7,18). As a result, NO group transfer is graded with the degree of hypoxia (6,19). Studies in mice with a genetic deficiency in SNO turnover demonstrate the existence of an apparent equilibrium of NO between Hb and glutathione in vivo and provide strong evidence that Snitrosothiols (SNOs) regulate vascular resistance (20).…”