Crystal structure of Root-effect hemoglobins

Some fish hemoglobins (Hbs) exhibit the Root effect, which produces a drastic reduction in oxygen-carrying capacity and cooperativity at acidic pH. So far two main hypotheses, essentially based on the stereochemical model (structural translation of the concerted model) [1], have been proposed to interpret the loss of the oxygen-driven, cooperative T-R transition due to the Root effect: 1) one invoking a destabilization of the R form at acidic pH, due to a cluster of positive charge at the β1β2 interface [2]; and 2) the other suggesting stabilization of the low affinity T state [3,4]. In order to investigate the influence of protonation on the structure of fish Hbs, we determined the crystal structures of the Antarctic fish Trematomus bernacchiii hemoglobins (HbTb) and of the catodic component of another Antarctic fish, Trematomus newnesi, Hb (HbCTn), both endowed with Root effect. The structure of HbTb deoxy state, previously solved at a moderate resolution [3], has been determined at pH values of 6.0 and 8.4 to a resolution of 1.30 and 1.78 Ǻrespectively. Furthermore, the structure of HbCTn has been determined to 2.0 Ǻ. Interestingly, no difference in the quaternary structure of HbTb is apparent at the two pH values investigated, and only few differences are present on their tertiary structure. Major differences are conformational changes in the CDα (His55α-Asp48α) and EFβ (Asp72β-His69β) corners of HbTb, where the lack of these salt bridges destabilizes HbTb in the deoxy state at basic pH. An important common structural feature of the three structures investigated is particularly relevant to the Root effect: the persistence of Asp95α1-Asp101β2 H-bond at the α1β2 interface in HbTb at pH up to 8.4, and in HbCTn. This result is twice surprising since 1) it requires an Asp pKa larger than 8.4 in HbTb, probably due to the assistance of a neighbor charged Asp99β2, and 2) despite the presence of hindrance and of potential hydrogen bonding competitors at the α1β2 interface, HbCTn shows the same motif of Asp95α1-Asp101β2 H-bond reported in HbTb. These findings strongly corroborates the hypothesis [3] that this Asp-Asp motif provides two of the protons released upon oxygenation of Root-effect Hbs within the pH range from 6.0 up to 8.4. Interestingly, HbTb does not presents the typical salt bridge His146β-Glu94β that accounts in the adult human Hb (where Glu94 is substituted by Asp) for the Bohr effect and in carp Hb for the Root effect, despite the side chain His146β and Glu94β are close enough to build the salt bridge and no strain is apparently generated in the AFHb structure. [1] Perutz, M.F., Fermi, G., Luisi, B., Shaanan, B., Liddington, R.C., Acc. Chem. Res. 1987, 20, 309. [2] Mylvaganam, S.E., Bonaventura, C., Bonaventura J., Getzoff, E.D.., Nature Str. Biol. 1996, 3, 275. [3] Ito,N., Komiyama, N.H., Fermi, G., J. Mol. Biol. 1995, 250 648. [4] Mazzarella, L., D’Avino, R., di Prisco, G., Savino, C., Vitagliano, L., Moody, P.C., Zagari, A. J. Mol. Biol. 1999, 287, 897.