## 9 Discussion
This fact poses the question of how erythrocytes can elicit the range of responses to hemichrome with such a small amount of metHb. We suggest that this can be achieved by intramolecular anion-induced nucleophilic displacement of molecular dioxygen via an intermediate ferrous heme/anion complex (a low-spin hemichrome) [20]. Such a reaction would follow the scheme:  
\[ \begin{array}{cccc}\delta+&\delta\text{-}\\ \text{Hb}_{\text{sub}}\text{Fe}(\text{II})\text{O}_{2}&+&\text{N-}(\text{His -E7})\\ \text{$k$N}\\ \text{$\longrightarrow$}&\text{metHb}_{\text{sub}}\text{Fe}(\text{III})\text{ (N-)}&+&\text{O}_{2}\text{-}\end{array} \]  
In this reaction, nucleophilic displacement can be caused within the heme pocket by the N\({}^{\text{e}}\)-nitrogen of the distal His (E7) instead of nucleophilic incursion of the water molecule or the hydroxyl ion from outside the molecule. As indicated above, hemichrome formation occurs at every stage during the autoxidation reaction of HbO\({}_{2}\) (_i.e._, at the initial, intermediate, and final stages) as a function of the pH and temperature of the solution. While the reaction proceeds along this scheme, hemichromes may be derived not only from HbO\({}_{2}\) species, but also from deoxyHb and metHb (Figure 21). Vital hemichromes in erythrocytes _in situ_ might arise from this intramolecular anion-induced nucleophilic displacement of O\({}_{2}\) via an intermediate ferrous heme/anion complex. Figure 21: Schematic representation of the role of the \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface in human HbO\({}_{2}\) A, redrawn from [PERSON] _et al._[24]. The \(\alpha_{1}\)-\(\beta_{1}\) (and \(\alpha_{2}\)-\(\beta_{2}\)) interface produces a conformational constraint in the constituted chains via tilting of the distal His (E7) residues so as to cause degradation of the Hb molecule to hemichrome, and subsequent Heinz-body clustering within the erythrocyte. In the spleen (which is responsible for the removal of aged and damaged red cells from the blood circulation), Heinz body-containing red cells become trapped and hence undergo hemolysis. By virtue of this endowment, Hb molecules control the fate (removal) of their own erythrocytes from the blood circulation depending on internal and extraneous circumstances, including pH and temperature. Our electron paramagnetic resonance (EPR) measurement study [17] showed a low-spin spectrum for the resulting oxidation products of isolated \(\beta\) chains with values of \(g_{1}=2.77\), \(g_{2}=2.27\), and \(g_{3}=1.68\), in addition to the usual aquo-met species with \(g\) values of 5.86 and 1.99 under a magnetic field of 0-500 mT at 8.0 K in 10 mM maleate buffer (pH 6.2) and in the presence of 50% (\(\
u\)/\(\
u\)) glycerol. According to [PERSON] _et al_. [11], such low-spin complexes characterized by the highest \(g\) values in the range 2.83-2.75 and the lowest \(g\) values in the range 1.69-1.63 have been designated as \"complex B\", indicating the crystal field parameters of the reversible hemichrome (_i.e._, a water-retained bis-histidine complex). The molar fraction of the hemichrome (complex B) in the oxidized \(\beta\) chains was estimated to be 85% at pH 6.2 because a low-spin species was in equilibrium with a high-spin species corresponding to the usual aquo-met species. Interestingly, recent crystallographic and EPR spectroscopic studies concerning tetrameric Hbs isolated from the Antarctic fish species _Trematomus bernacchi_, _Trematomus newnesi_, and _Gymnodraco acuticep_ show that endogenous coordination at the sixth coordination site of the heme iron could be the bis-histidyl adducts in the ferric state in the solid and solution state [26,76-78]. In Antarctic fish, isolated Hbs are readily oxidized at room temperature to a partial hemichrome state in which only the iron of the \(\beta\) chain is bonded to the distal His.