Human oxyhemoglobin showed a biphasic autoxidation curve containing two rate constants, i.e. k f for the fast autoxidation due to the ␣ chains, and k s for the slow autoxidation of the ␤ chains, respectively. Consequently, the autoxidation of the HbO 2 tetramer produces two different curves from the pH dependence of k f and k s . The analysis of these curves revealed that the ␤ chain of the HbO 2 tetramer does not exhibit any protoncatalyzed autoxidation, unlike the ␣ chain, where a proton-catalyzed process involving the distal histidine residue can play a dominant role in the autoxidation rate. When the ␣ and ␤ chains were separated from the HbO 2 tetramer, however, each chain was oxidized much more rapidly than in the tetrameric parent. Moreover, the separated ␤ chain was recovered completely to strong acid catalysis in its autoxidation rate. These new findings lead us to conclude that the formation of the ␣ 1 ␤ 1 contact produces in the ␤ chain a conformational constraint whereby the distal histidine at position 63 is tilted away slightly from the bound dioxygen, preventing the proton-catalyzed displacement of O 2. by a solvent water molecule. The ␤ chains have thus acquired a delayed autoxidation in the HbO 2 tetramer.The reversible and stable binding of molecular oxygen to the heme iron(II) is the basis of hemoglobin function. Consequently, much attention has been directed to the elucidation of the molecular mechanism of cooperative oxygen binding to the hemoglobin tetramer (1). However, the oxygenated form of hemoglobin, as well as of myoglobin, is known to be oxidized easily to the ferric(III) met form, which cannot bind molecular oxygen and is therefore physiologically inactive, with generation of the superoxide anion (2-5).To this autoxidation reaction, it has been widely accepted that hemoglobin is much more resistant as compared with myoglobin. Unlike myoglobin, Mansouri and Winterhalter (6) reported that oxyhemoglobin (HbO 2 ) showed a biphasic autoxidation reaction with a fast and a slow component. They also demonstrated that the ␣ chain was oxidized more rapidly than the ␤ chain in hemoglobin tetramer. At the same time, however, there have been a number of reports that such a rate difference was not observed between the ␣ and ␤ chains in the autoxidation reaction of HbA (7-9). Rather, Zhang et al. (9) showed that the rate of autoxidation was markedly enhanced when the HbO 2 tetramer dissociates into ␣␤ dimers. To clarify these discrepancies, we have recently examined systematically the effect of hemoglobin concentration on the autoxidation rate at several different values of pH, and found that human HbO 2 exhibits a biphasic autoxidation curve only in the pH range from neutral to acidic (5). By dissociation of tetramers into ␣␤ dimers, the rate of autoxidation for the fast component (due to the ␣ chain) was also found to increase markedly at the acidic pH, but the addition of 2,3-diphosphoglyceric acid offered no significant effect on the increment of the autoxidation rate (5).In the present pape...

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The oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met-form with generation of the superoxide anion. To make clear the possible role(s) of the distal histidine (H64) residue in the reaction, we have carried out detailed pH-dependence studies of the autoxidation rate, using some typical H64 mutants of sperm whale myoglobin, over the wide range of pH 5±12 in 0.1 m buffer at 25 8C. Each mutation caused a dramatic increase in the autoxidation rate with the trend H64V $ H64G $ H64L q H64Q . H64 (wild-type) at pH 7.0, whereas each mutant protein showed a characteristic pH-profile which is essentially different from that of the wild-type or native sperm whale MbO 2 . In particular, all the mutants have lost the acid-catalyzed process that can play a dominant role in the autoxidation reaction of most mammalian myoglobins or hemoglobins. Kinetic analyses of various types of pH-profiles lead us to conclude that the distal histidine residue can play a dual role in the nucleophilic displacement of O 2 2 from MbO 2 or HbO 2 in protic, aqueous solution. One is in a proton-relay mechanism via its imidazole ring, and the other is in the maximum protection of the FeO 2 center against a water molecule or an hydroxyl ion that can enter the heme pocket from the surrounding solvent.Keywords: dioxygen; heme oxidation; acid catalysis; distal (E7) residue; myoglobin mutants (sperm whale).The reversible and stable binding of oxygen to the heme iron(II) is the basis of myoglobin(Mb) and hemoglobin(Hb) functions. During reversible oxygen binding, however, the oxygenated form of myoglobin or hemoglobin is oxidized easily to the ferric met-form with generation of the superoxide anion [1]. Thus, stability property of the oxygenated form is of particular importance in vivo, as the iron(III) species cannot bind molecular oxygen and is therefore physiologically inactive. In this autoxidation reaction, the most remarkable feature is that the observed rate constant increases rapidly with increasing hydrogen ion concentration, although its pH-dependence curve is significantly different among the proteins from various sources [2±4]. In the case of human oxyhemoglobin (HbO 2 ), the reaction is the same but proceeds in a different manner. The separated a and b chains are both oxidized more rapidly than myoglobin molecules, but the HbO 2 tetramer turns to be quite resistant to autoxidation with the a and b chains being nonequivalent in the oxidation rate [5]. These recent new findings lead us to conclude that the formation of the a1b1 and a2b2 contacts produces a conformational constraint in the b chain whereby the distal (E7) histidine is tilted away slightly from the bound dioxygen, so as to prevent the proton-catalyzed displacement of O 2 2 from the FeO 2 center. The b chains have thus delayed remarkably the autoxidation rate of the HbO 2 tetramer [6]. To investigate the possible role(s) of the distal histidine residue in the autoxidation reaction, Brantley et al. [7] used site-directed mutagenesis of sperm whale and pi...