## 9 Discussion
A generalized pathway for this S\({}_{\text{N}}\)2 mechanism can be written using Mb (MbO\({}_{2}\)) as an example:  
\[\begin{array}{ccccc}&\delta+&\delta-&&\\ \text{MbFe}(\text{II})&\text{O}_{2}&+&\text{N}^{-}&\rightarrow&\left[\begin{array} []{c}\text{MbFe}\\ \text{O}_{2}\end{array  
pyridine was always involved in its rate equation in a complicated manner. By numerical calculations, however, it follows that oxyheme autoxidation can proceed with a rate constant that is much higher than 1 s\({}^{-1}\) in 0.1 M buffer, pH 8.5, at 25 \({}^{\circ}\)C (Figure 19). If such an oxyheme is placed in a protein matrix, it would be protected against the nucleophilic attack of the solvent water molecule or hydroxyl ion so as to reduce its autoxidation by a factor of \(>\)10\({}^{3}\). This was the case in our study with denatured MbO\({}_{2}\) in 8 M urea (the details of which can be referred to in [PERSON] _et al._[37]). A globin moiety can act as a \"breakwater\" in aqueous media even in denatured conditions with 8 M urea. Furthermore, if an oxyheme is embedded in the native Mb architecture, MbO\({}_{2}\) acquires remarkable stability against oxyheme autoxidation by a factor of \(\sim\)10\({}^{6}\) (Figure 19).  
With regard to oxyheme stability in human HbO\({}_{2}\), the half-life (\(t_{1/2}\)) was only 1.5 d in 0.1 M buffer and at physiological pH and temperature [16] even though the content of metHb in normal red cells _in situ_[1, 2] has been maintained to be \(\leq\)1% as a consequence of a NADH-dependent reducing system that can reduce the ferric-metHb resulting from autoxidation to deoxy-ferrous Hb [3-5]. However, as compared with the rates of monomeric mammalian Mbs, human HbO\({}_{2}\) seemed to be 2.2-3.6 times more stable against autoxidation (probably due to its tetrameric architecture). Even though the autoxidation rate is a function of pH and temperature, the observed first-order rate constant was 0.0023 h\({}^{-1}\) (\(t_{1/2}\) = 12.6 d) for the human HbO\({}_{2}\) autoxidation under the conditions of 0.1 M buffer at pH 7.2 and 25 \({}^{\circ}\)C in our study [10], because this value was calculated from the first rapid phase. Conversely,  
Figure 19: Roles of a globin moiety in Mb or Hb molecules, redrawn from [PERSON] _et al._[37].  
the relevant values of the monomeric mammalian Mbs were 0.0082 h\({}^{-1}\) (\(t_{1/2}\) = 3.5 d) for human MbO\({}_{2}\), 0.0072 h\({}^{-1}\) (\(t_{1/2}\) = 4.0 d) for bovine MbO\({}_{2}\), and 0.0050 h\({}^{-1}\) (\(t_{1/2}\) = 5.8 d) for sperm whale MbO\({}_{2}\) under the same conditions of human HbO\({}_{2}\).  
As mentioned above, the tetrameric human HbO\({}_{2}\) showed biphasic autoxidation curve in acidic solutions in terms of the -ln([HbO\({}_{2}\)]/[HbO\({}_{2}\)]\({}_{0}\)) _versus_ time plot; an initial rapid reaction (\(k_{\Lambda}^{\sslash}\)) could be followed with the slower second phase (\(k_{\Lambda}^{\sslash}\)). The difference in the rates between \(k_{\Lambda}^{\sslash}\) and \(k_{\Lambda}^{\sslash}\) decreased with increasing pH value of solutions, and finally disappeared (_i.e._, \(k_{\Lambda}^{\sslash}\)= \(k_{\Lambda}^{\sslash}\)) if the pH of the solutions was \(>\)8. A plot for the obtained values of \(k_{\Lambda}^{\sslash}\)/\(k_{\Lambda}^{\sslash}\)_versus_ pH indicated that the reaction involves a single dissociation group of amino-acid residue with pK\({}_{\mathrm{A}}\) = 7.4 (at 37 \({}^{\circ}\)C), probably a His residue. In addition, study of rate measurement for the isolated \(\alpha_{p\mathrm{-}\mathrm{M}\mathrm{B}}\) and \(\beta_{p\mathrm{-}\mathrm{M}\mathrm{B}}\) chains (and \(\alpha_{\mathrm{S}\mathrm{H}}\) and \(\beta_{\mathrm{S}\mathrm{H}}\) chains) disclosed that once the constituted chains were isolated from the parent molecule, both separated chains could be oxidized much more rapidly to the ferric met-form over the measured pH range (5-10) when compared with the respective rates (\(k_{\Lambda}^{\sslash}\) and \(k_{\Lambda}^{\sslash}\)) of the parent molecules calculated from biphasic autoxidation curves as a reference, and that there could be no practical differences regarding the rate between the isolated individual chains. These findings may mean that the individual chains have acquired considerable resistance to oxyheme autoxidation in their tetrameric protein architecture. It was also suggested that human HbO\({}_{2}\) seems to have acquired a pH-sensitive molecular device as a consequence of development of the tetrameric protein architecture. Hence, the molecule shows remarkable stability against oxyheme autoxidation more than in cases of isolated individual chains from the parent molecules and monomeric mammalian Mbs. Moreover, it was evident that only two valency hybrids, _i.e._, (\(\alpha^{3+}\beta^{2+}\))\({}_{2}\) and (\(\alpha^{2+}\beta^{3+}\))\({}_{2}\), emerged in the tetrameric HbO\({}_{2}\) autoxidation even though, in theory, seven species of valency hybrids can be in existence in theory during the reaction time course: (\(\alpha^{2+}\alpha^{2+}\beta^{3+}\beta^{2+}\)), (\(\alpha^{2+}\beta^{3+}\))\({}_{2}\), (\(\alpha^{2+}\beta^{3+}\))\({}_{2}\), (\(\alpha^{3+}\beta^{2+}\))\({}_{2}\), (\(\alpha^{2+}\alpha^{3+}\beta^{2+}\))\({}_{2}\), (\(\alpha^{2+}\alpha^{3+}\beta^{2+}\))\({}_{2}\), (\(\alpha^{2+}\alpha^{3+}\beta^{3+}\beta^{3+}\)) and (\(\alpha^{3+}\alpha^{3+}\beta^{3+}\beta^{2+}\)). Computer simulations on the basis of spectrophotometric, isoelectric-focusing electrophoretic and polyacrylamide gel electrophoretic measurements indicated that \(k_{\Lambda}^{\sslash}\) represents intrinsic \(\alpha\)-chain oxidation and \(k_{\Lambda}^{\sslash}\) reflects intrinsic \(\beta\)-chain oxidation. These findings led us to conclude that the \(\beta\) chain manifests delayed autoxidation in human HbO\({}_{2}\), which could be much more evident in acidic solutions.  
**Figure 20.** Schematic representation of the role of the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface in human HbO\({}_{2}\), redrawn from [PERSON] _et al._[17]. The \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface produces in the \(\beta\) chain a tilting of the distal (E7) His residue, thereby preventing the proton-catalyzed displacement of O\({}_{2}^{-}\) by a solvent water molecule.  
Figure 20 shows our proposal with respect to this issue as reported in 1998 [17] and 2002 [18] in which we suggested a new function to the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface for stabilizing the HbO\({}_{2}\) tetramer against acidic autoxidation. That is, the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface produces a conformational constraint in the \(\beta\) chain whereby the distal (E7) His at position 63 is tilted slightly away from the bound O\({}_{2}\) so as to prevent the proton-catalyzed displacement of O\({}_{2}^{-}\) from the Fe(II)O\({}_{2}\) center by entrance of a water molecule. The \(\beta\) chain thus acquires a remarkably delayed oxidation rate in the HbO\({}_{2}\) tetramer, and this is the origin of the chain heterogeneity seen in acidic solutions of HbO\({}_{2}\) autoxidation.  
In fact, the most recent refinement of the crystallographic structure of human HbO\({}_{2}\) (where the O\({}_{2}\) molecule is clearly visible in the high-resolution electron density maps) not only demonstrated that the hydrogen bond made by His E7\(\beta\) (_i.e._, \(\beta\)63 His) is much weaker than that made by His E7\(\alpha\) (_i.e._, \(\alpha\)58 His), but also substantiated that the geometry of the ligand and distal histidine is slightly different in the two subunits, with the O\({}_{2}\) atom lying \(2.7\pm 0.1\) A (10\({}^{-1}\) nm) from the N\({}^{e}\) atom of the distal histidine in the \(\alpha\) subunits, and 3.0 A away in the \(\beta\) subunits [66]. However, it is an unavoidable fact that negligible changes are found for the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface with respect to examination of the crystal structure.  
Similar phenomena suggesting participation of the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface (_i.e._, participation of the inherent tilting capability of the distal (E7) His residues of the Hb molecule) garnered our interest. As shown in Figures 5 and 6, our UV/VIS spectroscopic study suggested that human HbO\({}_{2}\) was highly susceptible to hemichrome formation even under physiological pH and temperature and, once isolated from the tetrameric parent molecule, the \(\alpha\) (\(\alpha_{\rho\text{-}\text{MB}}\)) and \(\beta\) (\(\beta_{\rho\text{-}\text{MB}}\)) chains showed much higher susceptibilities to hemichrome when compared with the parent molecule.  
Heinz bodies are intra-erythrocytic inclusions of hemichrome formed as a result of Hb oxidation. They have been detected and characterized in drug-induced hemolytic anemia, defects in the intra-erythrocytic reducing system (e.g., G-6-PD deficiency) and in unstable Hb disease [21,22]. Using unstable Hb disease as an example, quite a large number of unstable Hbs have been reported. Regarding the molecular pathogenesis of unstable Hbs, it is known that the instability of labile Hb variants in patients can be attributed to amino-acid substitutions (or deletions), which disrupt and perturb the Hb structure via interference with \(\alpha\)-helix formation, disruption of heme binding, or altered \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) or \(\alpha_{1}\)-\(\beta_{2}\) (and \(\alpha_{2}\)-\(\beta_{1}\)) contacts [21]. The consequent changes in circulating red cells in patients with unstable Hb disease include: an inherent tendency towards irreversible denaturation of Hb or globin due to a defect in the amino-acid composition of labile Hb molecules; a continuous tendency toward hemichrome formation; precipitation or aggregation of Hb molecules resulting in buildup of the molecules to form Heinz bodies; and hemolysis.  
Exposure of red cells to acetylcholinesterasehydrazine and subsequent staining with crystal violet also revealed a greater abundance of Heinz bodies within G-6-PD-deficient cells compared with that in normal cells [67,68]. In addition, [PERSON] _et al._[69] and [PERSON] [70] reported that Heinz bodies often appeared in normal-aging red cells, and that this age-related appearance of Heinz bodies was particularly pronounced in splenectomized individuals [71]. According to several authors [72-74], aged or damaged red cells affected by drugs may be \"filtered off\" by the spleen irrespective of whether or not they contain Heinz bodies in a similar manner to the filtering off of red blood cells in patients with unstable Hb caused by unstable Hb hemolytic anemia.  
Using freshly drawn venous blood from healthy donors, we found that the number of Heinz bodies formed in red cells increased with increasing temperature when blood samples were subjected to mild heating at \(>\)37 \({}^{\circ}\)C for 30 min [23,24]. Under identical conditions of mild heating, we measured blood fluidity using a MC-FAN [24] because it involves a characteristic V-shaped groove array with micro flow paths (width, 7 \(\upmu\)m; length, 30 \(\upmu\)m; depth, 4.5 \(\upmu\)m) engraved on a single-crystal silicon substrate in an integral circuit. When red cells passing through individual micro-channel arrays were monitored using an inverted metallographic microscope, images revealed good erythrocyte deformability when temperature-untreated samples were subjected to MC-FAN. Conversely, a marked decline in erythrocyte deformability was observed in blood samples treated at temperatures \(>\)37 \({}^{\circ}\)C for 30 min. Temperature-treated samples also demonstrated an increased transit time for low transit sample volumes. We therefore concluded that erythrocyte deformability decreased with increasing temperature of blood samples at \(>\)37 \({}^{\circ}\)C. The combination of our experimental findings and _in situ_ observations suggests that instability leading to hemichrome formation is not only a peculiarity of labile Hb variants, but is also an innate characteristic of physiologically normal Hb molecules. In this regard, one must note that monomeric bovine heart MbO\({}_{2}\) did not show a propensity for hemichrome formation over a wide range of pH (4.5-10.5) and temperature from physiological to temperatures just before thermal unfolding [20].  
In our experiments, the occurrence of hemichrome can be described as follows:  
\[k_{\mathrm{A}}\] \[\mathrm{Hb(II)O_{2}\to metHb(III)+4(O_{2}\text{-})\to hemichrome} \tag{13}\]  
Taking into account the accepted framework of the S\({}_{\mathrm{N}}\)2 mechanism for autoxidation [59-63] and the accepted framework for hemichrome formation [11] in physiologically conditions, hemichrome can form as follows:  
\[\mathrm{Hb_{subFe}(II)O_{2}\quad+\quad OH\text{-}(or\ H_{2}O)\quad\longrightarrow \left[\begin{array}{c}\par\par\par\par\par  
The content of metHb in normal erythrocytes [1,2] has been reported to be \(\leq\)1% by a NADH-dependent enzyme system [3-5].