Biochemical Characterization of HumanS-Nitrosohemoglobin: EFFECTS ON OXYGEN BINDING AND TRANSNITROSATION

Rakesh P Patel,Neil Hogg,Netanya Y Spencer,B Kalyanaraman,Sadis Matalon,Victor M Darley-Usmar

doi:10.1074/jbc.274.22.15487

Abstract

S-Nitrosation of cysteine beta93 in hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in vivo, and transnitrosation reactions of deoxygenated SNO-Hb are proposed as a mechanism leading to release of NO and control of blood flow. However, little is known of the oxygen binding properties of SNO-Hb or the effects of oxygen on transnitrosation between SNO-Hb and the dominant low molecular weight thiol in the red blood cell, GSH. These data are important as they would provide a biochemical framework to assess the physiological function of SNO-Hb. Our results demonstrate that SNO-Hb has a higher affinity for oxygen than native Hb. This implies that NO transfer from SNO-Hb in vivo would be limited to regions of extremely low oxygen tension if this were to occur from deoxygenated SNO-Hb. Furthermore, the kinetics of the transnitrosation reactions between GSH and SNO-Hb are relatively slow, making transfer of NO+ from SNO-Hb to GSH less likely as a mechanism to elicit vessel relaxation under conditions of low oxygen tension and over the circulatory lifetime of a given red blood cell. These data suggest that the reported oxygen-dependent promotion of S-nitrosation from SNO-Hb involves biochemical mechanisms that are not intrinsic to the Hb molecule.

Highlights

S-Nitrosation of cysteine ␤93 in hemoglobin (S-nitrosohemoglobin (SNO-Hb)) occurs in vivo, and transnitrosation reactions of deoxygenated SNO-Hb are proposed as a mechanism leading to release of NO and control of blood flow
Our results demonstrate that SNO-Hb has a higher affinity for oxygen than native Hb. This implies that NO transfer from SNO-Hb in vivo would be limited to regions of extremely low oxygen tension if this were to occur from deoxygenated SNOHb
Nitric oxide is synthesized in endothelial cells via the oxidation of L-arginine, a reaction catalyzed by the type III nitric-oxide synthase and stimulated by physiological processes such as blood flow over the endothelium [4]

Summary

EFFECTS ON OXYGEN BINDING AND TRANSNITROSATION*

(Received for publication, November 19, 1998, and in revised form, March 10, 1999). Rakesh P. Since the formation of SNO-Hb represents a physiologically relevant Hb derivative in which Cys-␤93 is modified, we have examined the effect of S-nitrosation of Hb on its oxygen binding properties It is not clear how the oxygen-sensitive mechanisms underlying conformation-dependent NO release function. We have examined the kinetics and equilibria of transnitrosation reactions between Hb and GSNO and fitted the results to a bimolecular reversible reaction These data show that S-nitrosation increases the oxygen affinity of Hb, ensuring that if a Hb deoxygenation-sensitive mechanism of NO release exists, SNO-Hb would only promote vasorelaxation at sites of very low oxygen concentrations, i.e. at sites where the Hb existed primarily in the deoxygenated state. These data are discussed in the context of SNO-Hb as a potential modulator of blood flow

EXPERIMENTAL PROCEDURES

Form of Hb

RESULTS

Allosteric effect

DISCUSSION