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In addition to interacting with hemoglobin as a heme ligand to form nitrosylhemoglobin, NO can react with cysteine sulfhydryl groups to form S-nitrosocysteine or cysteine oxides such as cysteinesulfenic acid. Both modes of interaction are very sensitive to the quaternary structure of hemoglobin. To directly view the interaction of NO with quaternary-T deoxyhemoglobin, crystallographic studies were carried out on crystals of deoxyhemoglobin that were exposed to gaseous NO under a variety of conditions. Consistent with previous spectroscopic studies in solution, these crystallographic studies show that the binding of NO to the heme groups of crystalline wild-type deoxyhemoglobin ruptures the Fe-proximal histidine bonds of the alpha-subunits but not the beta-subunits. This finding supports Perutz's theory that ligand binding induces tension in the alpha Fe-proximal histidine bond. To test Perutz's theory, deoxy crystals of the mutant hemoglobin betaW37E were exposed to NO. This experiment was carried out because previous studies have shown that this mutation greatly reduces the quaternary constraints that oppose the ligand-induced movement of the alpha-heme Fe atom into the plane of the porphyrin ring. As hypothesized, the Fe-proximal histidine bonds in both the beta- and the alpha-subunits remain intact in crystalline betaW37E after exposure to NO. With regard to S-nitrosocysteine or cysteine oxide formation, no evidence for the reaction of NO with any cysteine residues was detected under anaerobic conditions. However, when deoxyhemoglobin crystals are first exposed to air and then to NO, the appearance of additional electron density indicates that Cys93(F9)beta has been modified, most likely to cysteinesulfenic acid. This modification of Cys93(F9)beta disrupts the intrasubunit salt bridge between His146(HC3)beta and Asp94(FG1)beta, a key feature of the quaternary-T hemoglobin structure. Also presented is a reanalysis of our previous crystallographic studies [Chan, N.-L., et al. (1998) Biochemistry 37, 16459-16464] of the interaction of NO with liganded hemoglobin in the quaternary-R2 structure. These studies showed additional electron density at Cys93(F9)beta that was consistent with an NO adduct. However, for reasons discussed in this paper, we now believe that this adduct may be the Hb-S-N.-O-H radical intermediate and not Hb-S-N=O as previously suggested.

Although numerous reports have documented that the S-nitrosylation of cysteine residues by NO alters the activities of a wide variety of proteins, the direct visualization and the structural consequences of this reversible modification have not yet been reported for any protein. Here we describe the crystal structure of S-nitroso-nitrosylhemoglobin determined at a resolution of 1.8 A. The specific reaction of NO with Cys93beta is confirmed in this structure, and a large S-nitrosylation-induced change in the tertiary structure of the COOH-terminal dipeptides of the beta subunits provides additional insight into the stereochemical mechanism by which blood flow is regulated by the interaction of NO with hemoglobin.