The enzymatic function of the honorary enzyme: S-nitrosylation of hemoglobin in physiology and medicine

Abstract

The allosteric transition within tetrameric hemoglobin (Hb) that allows both full binding to four oxygen molecules in the lung and full release of four oxygens in hypoxic tissues would earn Hb the moniker of 'honorary enzyme'. However, the allosteric model for oxygen binding in hemoglobin overlooked the essential role of blood flow in tissue oxygenation that is essential for life (aka autoregulation of blood flow). That is, blood flow, not oxygen content of blood, is the principal determinant of oxygen delivery under most conditions. With the discovery that hemoglobin carries a third biologic gas, nitric oxide (NO) in the form of S-nitrosothiol (SNO) at β-globin Cys93 (βCys93), and that formation and export of SNO to dilate blood vessels are linked to hemoglobin allostery through enzymatic activity, this title is honorary no more. This chapter reviews evidence that hemoglobin formation and release of SNO is a critical mediator of hypoxic autoregulation of blood flow in tissues leading to oxygen delivery, considers the physiological implications of a 3-gas respiratory cycle (O₂/NO/CO₂) and the pathophysiological consequences of its dysfunction. Opportunities for therapeutic intervention to optimize oxygen delivery at the level of tissue blood flow are highlighted.

Keywords: Allostery; Erythrocyte; Hypoxic vasodilation; Oxygen delivery; S-Nitroso-hemoglobin; S-Nitrosothiol.

Figure 1.

Role of allostery in hypoxic vasodilation. SNO-Hb mediates tissue blood flow increase in hypoxia in proportion to Hb deoxygenation (see also Figure 3). A. Limb blood flow increases linearly with decreasing oxygen saturation of the perfusing blood. Adapted from (Ross et al., 1962). B. Oxygenation-state allostery controls the reactivity of SNO-Hb. Isolated SNO-Hb in the oxygenated R-state is stable, but is destabilized and reactive in the in hypoxic T-state; loss of SNO from T-state Hb is potentiated by glutathione (GSH) serving as a SNO acceptor. Adapted from (Jia et al., 1996). C. SNO-Hb induces hypoxic vasodilation. Isolated SNO-Hb produces dose-dependent contraction of isolated aorta in an organ bath under room air (21% oxygen, black) but vasodilation under hypoxia (1% oxygen, red). Adapted from (McMahon et al., 2000). D. Human RBCs dilate blood vessels in proportion to Hb desaturation. Adapted from (McMahon et al., 2002).

Figure 2.

Hb as a SNO-generating enzyme. I. Converting FeNO to SNO through SNO synthase activity (A-D). A. Absorbance spectroscropy of Hb-bound FeNO as a function of oxygen (room air) added to the sample, comparing recombinant human Hb and the Cys93Ala mutant, showing that the Cys93Ala mutant lacks a ‘plateau phase’ (from ^~15–45 ul) representing loss of FeNO in production of SNO, whereas the wildtype Hb demonstrates O₂-dependent displacement of NO from heme. Adapted from (Gow and Stamler, 1998). B. Electron Paramagnetic Resonance (EPR) measurements of nitrite/Hb under deoxy (black)/oxy (red)/deoxy (blue) cycling. FeNO Hb (black) produced from nitrite under deoxy conditions; loss of this FeNO spectrum upon oxygenation (red), consistent with transfer of NO from heme to Cys to form SNO (note: a newly formed Cys radical is observed; red); and regeneration of FeNO under deoxygenation (blue), consistent with release of NO from SNO upon return to the deoxygenated state (blue), as described by (Pezacki et al., 2001). Adapted from (McMahon et al., 2002). C. Absorbance spectroscropy of metHb over time under deoxygenated conditions in the presence of NO, demonstrating increase in Fe(II)-nitrosyl Hb (heme-Fe(II)NO), and decrease in Fe(III) nitrosyl Hb (heme-Fe(III)NO), indicative of reductive nitrosylation via heme-Fe(III) that precedes SNO formation. Contemporaneous measurements confirm the formation of SNO-Hb. Adapted from (Luchsinger et al., 2003). D. Hb is SNO synthase. Treatment of Hb by cycling between deoxygenated state to oxygenated state in the presence of nitrite leads to near-100% formation of SNO-Hb. Adapted from (Angelo et al., 2006). II. Hb is a transnitrosylase. E. Treatment of deoxygenated RBCs with 1 μM NO (to create FeNO) and subsequent oxygenation (to promote SNO-Hb formation in cytosol) followed by deoxygnation promotes transfer of SNO to membrane-assocated proteins (including AE-1). Adapted from (Pawloski et al., 2001).

Figure 3.

SNO(Cys93)-Hb induces hypoxic vasodilation and tissue oxygen delivery. A. RBCs derived from C93A mutant animals have diminished SNO-Hb (Alfred Hausladen,JSS unpublished data). B. RBCs derived from C93A mutant animals cannot effectively dilate blood vessels under hypoxia. C. Mice bearing RBCs with wildtype human Hb (C93) exhibit higher muscle oxygen level (pO2) compared to mice bearing mutant Hb unable to carry SNO at Cys93 (Cys93Ala). D. Mice bearing RBCs with mutant human Hb (C93) exhibit impaired autoregulation of blood flow in peripheral limbs. Panels B-D adapted from (Zhang et al., 2015).

Figure 4.

Clinical manifestations of SNO-Hb function. A. Arterial-venous difference in SNO-Hb content in normal humans breathing room air. SNO-Hb is high in oxygenated arterial blood and lower in deoxygenated venous blood (protected from air) consistent with allosteric regulation of SNO bioactivity. Adapted from (McMahon et al., 2002). B. Mice lacking βCys93 SNO-Hb exhibit elevated rate of death after myocardial infarction consequent upon ischemia-reperfusion injury. Adapted from (Zhang et al., 2016). C. Tissue oxygenation is elevated in sheep transfused with 2 units of blood renitrosylated by treatment with ethyl nitrite vs blood left untreated and deficient in SNO-Hb. Adapted from (Reynolds et al., 2013). D. Reduced complications in pediatric bypass patients after transfusion correlates with patient SNO-Hb level post-transfusion. Adapted from (Matto et al., 2018).