Origin and mechanism of thermal insensitivity in mole hemoglobins: a test of the 'additional' chloride binding site hypothesis

The structural and evolutionary origins underlying the effect of temperature on the O-2 binding properties of mammalian hemoglobins (Hbs) are poorly understood, despite their potential physiological importance. Previous work has shown that the O-2 affinities of the blood of the coast mole (Scapanus orarius) and the eastern mole (Scalopus aquaticus) are significantly less sensitive to temperature changes than that of the star-nosed mole (Condylura cristata). It was suggested that this difference may arise from the binding of 'additional' chloride ions within a cationic pocket between residues 8His, 76Lys and 77Asn on the beta-like delta-globin chains of coast and eastern mole Hbs. To test this hypothesis, we deduced the primary sequences of star-nosed mole and American shrew mole (Neurotrichus gibbsii) Hb, measured the sensitivity of these respiratory proteins to allosteric effector molecules and temperature, and calculated their overall oxygenation enthalpies (Delta H'). Here we show that the variability in Delta H' seen among mole Hbs cannot be attributed to differential Cl- binding at delta 8, delta 76 and delta 77, as the Cl- sensitivity of mole Hbs is unaffected by amino acid changes at this site (i.e. the proposed 'additional' Cl- binding site is not operational in mole Hbs). Rather, we demonstrate that the numerically low Delta H' of coast and eastern mole Hbs results from heightened proton binding relative to other mole Hbs. Comparative sequence analysis and molecular modelling moreover suggest that this attribute evolved in a common ancestor of these two fossorial lineages and arises from the development of a salt bridge between a pair of amino acid residues (delta 125His and alpha 34Glu/Asp) that are not present in other mole Hbs.