Combined Raman and X-ray crystallography reveals multiple coordinations and quaternary states of fish hemoglobins

Many fish hemoglobins (Hbs) are endowed with Root effect, that produces a decrease in oxygen affinity at low pH, associated with complete loss of cooperativity. Over the years, several hypotheses on the structural determinants of the Root effect have been suggested. Up to now, all the structural explanations of the Root effect have been based on the two-state model, in which the Root effect is related to an increase of the allosteric equilibrium between the R and T state at acidic pH [1-3]. Here, we report the crystal structures of the deoxy and carbomonoxy forms of the non-Root-effect major component Hb 1 isolated from the Antarctic fish (Af) Trematomus newnesi (Hb1Tn). Surprisingly, in the deoxy state of the non-Root effect Hb1Tn, the inter-aspartic hydrogen bond between Asp95α and Asp101β at the α1β2 interface, which is believed to be important in Root effect [2], is observed. A combined Resonance Raman / x-ray crystallography of this AfHb has revealed heterogeneity in the deoxy coordination. Novel ligated states are observed in both a T-like state and a R/T intermediate quaternary state. Three of four independent CO coordination states are not assisted by the hydrogen bond of the distal histidyl, that swings out of the heme pocket. These un-assisted CO coordination states (supported by FT-IR spectroscopy solution studies) are associated with unusually small thermal fluctuations which characterise both α and β CD corners. The accessibility of ligated states within three different quaternary structures (T, R and R/T intermediate [4]) suggests a novel structural explanation of protein allostery, based on a three-state Edelstein’s model [5]. Grant Sponsors: PRIN , PNRA. [1] Mazzarella et al. Proteins, 2006, 65(2), 490-498. [2] Mazzarella et al. Proteins, 2006, 62(2), 316-321 [3] Yokoyama et al. J. Biol. Chem. 2004, 279, 28632-28640. [4] Vitagliano et al. J. Am. Chem. Soc., 2008, 130, 10527-10535. [5] Edelstein, J. Mol. Biol., 1996, 256, 737-744