BIS-HISTIDYL COORDINATION IN TETRAMERIC HEMOGLOBINS: COLD-ADAPTED FISH HEMOGLOBINS VS HUMAN HEMOGLOBIN

All tetrameric hemoglobins from the Antarctic fish, included Trematomus bernacchii, HbTb, in its ferric state promptly, and distinctively from all the other tetrameric hemoglobins, form a mixture of aquo-met at the α subunits and bis-histidyl adduct (hemichrome) at the β subunits 1,2. The role of the tertiary and quaternary structure in the hemichrome formation in HbTb is still unknown. Here we report the cloning, expression, purification, spectroscopic and computational characterization of the β-chain of HbTb (Tbβ), along with a novel cystallographic determination of the ferric β-chain of human Hb (β4-HbA). As β4-HbA 3,4, Tbβ self assembles to form a β4 homotetramer, but, differently from β4-HbA, Tbβ forms quantitatively a reversible ferric bis-histidyl adduct in the ferric state. Indeed, the herein presented crystal structure of the ferric β4-HbA hosts an aquo-met coordination in all the four independent chains, and not hemichrome. A molecular dynamics study on the isolated β-subunit of HbTb indicates that the ability to form hemichrome is an intrinsic feature of Tbβ chain, probably due to the high intrinsic flexibility associated with its sequence. Differently from HbTb α2β2 heterotetramer 5,6, in the ferrous state Tbβ forms a bis-histidyl adduct (hemochrome). On the bases of these experimental, crystallographic and computational results, a discussion on the effect of quaternary structure on the stability of endogenous ferrous and ferric hexa-coordination is presented. We acknowledge PNRA and PRIN for financial support (1) Merlino, A.; Vitagliano, L.; Howes, B.; Verde, C.; di Prisco, G.; Smulevich, G.; Sica, F.; Vergara, A. Biopolymers 2009, 91, 1117. (2) Vergara, A.; Franzese, M.; Merlino, A.; Vitagliano, L.; di Prisco, G.; Verde, C.; Lee, H. C.; Peisach, J.; Mazzarella, L. Biophys. J. 2007, 93, 2822. (3) Rachmilewitz, E. A.; Peisach, J.; Blumberg, W. E. J Biol Chem 1971, 246, 3356. (4) Shikama, K. Chem. Rev. 1998, 98, 1357. (5) Vergara, A.; Vitagliano, L.; Merlino, A.; Sica, F.; Marino, K.; Verde, C.; di Prisco, G.; Mazzarella, L. J Biol Chem 2010, 285, 32568. (6) Mazzarella, L.; Vergara, A.; Vitagliano, L.; Merlino, A.; Bonomi, G.; Scala, S.; Verde, C.; di Prisco, G. Proteins 2006, 65, 490.