Reaction of S-Nitrosoglutathione with the Heme Group of Deoxyhemoglobin

Abstract

The mechanism of interaction between S-nitrosoglutathione (GSNO) and hemoglobin is a crucial component of hypotheses concerning the role played by S-nitrosohemoglobin in vivo. We previously demonstrated (Patel, R. P., Hogg, N., Spencer, N. Y., Kalyanaraman, B., Matalon, S., and Darley-Usmar, V. M. (1999) J. Biol. Chem. 274, 15487-15492) that transnitrosation between oxygenated hemoglobin and GSNO is a slow, reversible process, and that the reaction between GSNO and deoxygenated hemoglobin (deoxyHb) did not conform to second order reversible kinetics. In this study we have reinvestigated this reaction and show that GSNO reacts with deoxyHb to form glutathione, nitric oxide, and ferric hemoglobin. Nitric oxide formed from this reaction is immediately autocaptured to form nitrosylated hemoglobin. GSNO reduction by deoxyHb is essentially irreversible. The kinetics of this reaction depended upon the conformation of the protein, with more rapid kinetics occurring in the high oxygen affinity state (i.e. modification of the Cys␤-93) than in the low oxygen affinity state (i.e. treatment with inositol hexaphosphate). A more rapid reaction occurred when deoxymyoglobin was used, further supporting the observation that the kinetics of reduction are directly proportional to oxygen affinity. This observation provides a mechanism for how deoxygenation of hemoglobin/myoglobin could facilitate nitric oxide release from Snitrosothiols and represents a potential physiological mechanism of S-nitrosothiol metabolism. The interaction between S-nitrosoglutathione (GSNO)1 and hemoglobin has become an area of intense interest due to the discovery that circulating erythrocytes contain measurable levels of S-nitrosohemoglobin (HbSNO) (1), and that the combination of S-nitrosohemoglobin and glutathione (GSH) will relax vessels in an oxygen sensitive manner (2). HbSNO is a posttranslationally modified form of hemoglobin that contains a nitrosated cysteinyl residue at the ␤-93 position. The mechanism for the formation of HbSNO in vivo remains unknown, and the functional consequences of this modification are the subject of intense debate (1-4). It has been suggested that S-nitrosation of Hb represents an oxygen-sensitive mechanism to control vascular tone (1). The crux of this hypothesis is that transnitrosation, i.e. the chemical transfer of the nitroso group, between Hb and GSH is an oxygen-sensitive reaction in which deoxygenation of Hb allows the eventual formation of GSNO, which may then act as a vasodilator. The mechanism by which intra-erythrocyte GSNO acts as a vasodilator is as yet unknown. In a new twist to this hypothesis it has recently been suggested that rapid transnitrosation to form GSNO would be fatal, due to a massive vasodilatory response upon deoxygenation of Hb (5). In order to avoid such an outcome the majority of the NO has been proposed to be autocaptured at the deoxygenated heme, to give nitrosyl hemoglobin (HbNO), in which nitric oxide is bound to the iron of ferrous hemoglobin. It has been additionally suggested that...

Timolol Modulates Erythrocyte Nitric Oxide Bioavailability

“…The lower GSNO concentration obtained suggest that it was consumed in the irreversible reaction with deoxygenated haemoglobin to originate glutathione, methahemoglobin and NO [24]. In turn the nitric oxide reduces oxyhemoglobin molecules and originates nitrate and metahemoglobin [25].…”

Effects of S-Nitrosation and Cross-Linking of Hemoglobin on Hypoxic Pulmonary Vasoconstriction in Isolated Rat Lungs

“…These data suggest that allosteric influences as induced by Hb deoxygenation on the release of NO from free SNO-Hb are limited and are consistent with previous reports on SNOHb and GSH interactions. 14,26 Although dissociation of Hb tetramers into dimers is a potential problem when working with dilute Hb solutions, the perfusate Hb concentration of 100 mol/L in the present study is sufficient to preclude this concern. 23 In addition, IHP reduces dimerization by stabilizing T state hemoglobin.…”

Heme Redox Properties of S-Nitrosated Hemoglobin A0 and Hemoglobin S

“…Moreover, there were no detectable NO-heme adducts present prior to dithionite treatment, indicating that little or no NO was trapped at the heme prior to dithionite treatment. As reported by Spencer et al (36), GSNO, if formed to a significant extent, could have generated some NO-heme as a result of its interactions with deoxy Hb.…”

“…4. This paradigm, in which met-Hb plays a significant role, is based on results reported herein for SH-modified forms of Hb A 0 (NEM-Hb A 0 , PDS-Hb A 0 , and SNO-Hb A 0 ), and CPA-Hb (SNO-CPA-Hb), as well as reports from the literature (3,6,17,36,42,48). Possible responses to low oxygen conditions are shown, although the physiological condition is rarely "free" of oxygen, and met-Hb levels are usually low.…”

“…Spencer et al (36) reported that the interaction of deoxy-Hb A 0 with S-nitrosated glutathione (GSNO) leads to the formation of NO, met-Hb A 0 , and glutathione (GSH) (Equation 3), followed by the formation of a heme-NO adduct (Hb-NO) (Equation 4).…”