Structure and function of tetrameric hemoglobins and their mutants at a molecular and cellular level.

• Main Author: Balsamo, Anna
• Format: Doctoral or Postdoctoral Thesis
• Language: Italian; English
• Published: 2011
• Subjects: Antarctic; Antarc*
• Online Access: http://www.fedoa.unina.it/8790/; http://www.fedoa.unina.it/8790/1/balsamo_anna_24.pdf; https://doi.org/10.6092/UNINA/FEDOA/8790

The present Ph.D. thesis has focused on tetrameric hemoglobins (Hbs), both recombinant and natural, both from human origin and Antarctic fish, using a multidisciplinary approach based on spectroscopic, crystallographic and computational techniques. In particular the main scope of the research has been the elucidation of two still unsolved problems in the chemistry of tetrameric Hbs: 1) the role of the bis-histidyl heme coordination in the Hb function and oxidation process and 2) the role of the tertiary and quaternary structure in the modulation of the Root effect (namely drop of oxygen affinity with loss of cooperativity at low physiological pH). The first topic has been mainly approached through a comparative experimental (spectroscopic and crystallographic) / computational study of the β-subunits of human hemoglobin (β-HbA) and of the recombinant β-subunits Hb from Antarctic fish Trematomus bernacchii (β-HbTb), whose heterotetramer, in the ferric state, forms a mixture of aquo-met at the α-subunits and bis-histidyl adduct at the β-subunits. Similarly to the human β-chains, β-HbTb self-assembles to form the homotetramer (β4-HbTb); however, the latter quantitatively forms reversible ferric and ferrous bis-histidyl adducts, which are only partially present in the human tetramer (β4-HbA). The molecular dynamics study of the isolated β-subunit of the two Hbs indicates that the ability to form hemichrome is an intrinsic feature of the chain; moreover, the greater propensity of β-HbTb to form the bis-histidyl adduct is probably linked to the higher flexibility of the CD loop region. These findings are in perfect agreement with the X-ray structure of β4-HbA in the ferric (solved in this thesis) that hosts only aquo-met coordination. The mechanism of hemichrome formation was also investigated by determining the x-ray crystal structure of the fully oxidized Hb from Antarctic fish Trematomus newnesi and by performing a Resonance Raman microscopy study on the Hb crystals from a sub-Antarctic fish (Eleginops maclovinus). ...