| Summary: | Antarctic fish hemoglobins (AFHbs) exhibit a peculiar oxidation process. Upon oxidation, these tetrameric proteins show a remarkable propensity to evolve toward the formation of hemichrome, a species in which both proximal and distal hystidines are bound to the heme iron [1,2]. The AFHb isolated from Trematomus bernacchii also shows a drastic reduction of oxygen affinity and cooperativity at acidic pH ( Root effect), ascribed to an overstabilization of the T quaternary structure [3]. On the other hand, the AFHbs isolated from Trematomus newnesi , which bears a very high sequence identity with the former Hb, has a nearly pH-independent oxygen affinity. The crystal structure of the oxidized form of these two AFHbs has shown that a) the a chains go to aquo-met, whereas the b chains form the hemichrome, b) the quaternary structure is intermediate between the canonical R and T states [1,2]. The EPR analysis of the ferric AFHbs at physiological pH reveals two distinct hemichromes (I, II) in solution [4]. Interestingly, the pH dependence of EPR spectra and of the X-ray crystal structure of ferric HbTb suggests a correlation between the hemichrome stability and the Root effect. Indeed, the high-resolution crystal structures of the oxidized form of the AFHbs isolated from T. bernacchii at pH 7.6 and 6.0 (1.5 and 1.8 Å, respectively) show different quaternary structures and different heme coordination: at pH 7.6 the hemoglobin is in the a-aquomet/b-hemichrome form ad its quaternary structure is intermediate between the R and T state, whereas at pH 6 both hemes are penta-coordinated and the hemoglobin adopts the T quaternary structure. The EPR spectra of Root effect Hbs confirm the presence of a penta-coordinated, unligated ferric state at pH 6. Altogether, these findings suggest that the hemichrome instability at acidic pH for Root effect Hbs is related to some incompatibility of the hemichrome in the b subunits within a T quaternary structure. Interestingly, ferric AFHbs reveal a high peroxidasic activity with respect ...
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