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

Abstract

SUMMARY The structural and evolutionary origins underlying the effect of temperature on the O2 binding properties of mammalian hemoglobins (Hbs) are poorly understood, despite their potential physiological importance. Previous work has shown that the O2 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 β-like δ-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 (ΔH′). Here we show that the variability in ΔH′ seen among mole Hbs cannot be attributed to differential Cl– binding at δ8, δ76 and δ77, 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 ΔH′ 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 (δ125His and α34Glu/Asp) that are not present in other mole Hbs.

Molecular and physicochemical characterization of hemoglobin from the high-altitude Taiwanese brown-toothed shrew (Episoriculus fumidus)

“…In this regard it is significant that the response of E. fumidus Hb to saturating concentrations of this organophosphate (Log P 50 (0.75 mM DPG ? 0.1 M Cl -) -Log P 50 (0.1 M Cl -) = 0.22; Table 2) is within the lower range of that reported for semi-aquatic and fossorial moles (0.23-0.26; Campbell et al 2010a;Jelkmann et al 1981;Signore et al 2012). Jelkmann et al (1981) speculated that the low interaction of European mole Hb with DPG results from changes in the b-type chain contacts, conferred by amino acid substitutions at helical positions A1 (d4Thr ?…”

“…an alternate Cl -binding site must be operating in browntoothed shrew Hb). We recently presented evidence supporting this same conclusion for talpid mole Hbs (Signore et al 2012), which share a close phylogenetic affinity with shrews. Finally, it is of note that the DH of stripped Episoriculus Hb above pH 8.3 (*-46 kJ mol -1 ; data not shown), where the Bohr effect of Hb should be abolished, is markedly lower than pig Hb (-67 kJ mol -1 ; Weber et al 1987) and human HbA (-59 kJ mol -1 ; Atha and Ackers 1974).…”

“…2; Table 1). Notably, this attribute is also shared by deer mice and talpid moles (Campbell et al 2010a;Storz et al 2010;Signore et al 2012), which indicates the presence of discrete non-overlapping DPG and Cl -binding sites in these species. In this regard it is interesting that the above noted human Gower II e77Asn ?…”

“…For each treatment, Bohr coefficients were calculated using P 50 measurements obtained within the pH range 6.8-7.3. The overall enthalpy of oxygenation (DH, kJ mol -1 O 2 ), corrected for the solubilization heat of O 2 (-12.55 kJ mol -1 ), was calculated following Signore et al (2012).…”

“…A potential candidate in this regard is a113 His (Fig. 3), which is also found in the Hbs of hedgehogs and moles, and whose Hbs also possess comparably high fixed-acid Bohr effects (-0.52 to -0.62;Campbell et al 2010a;Jelkmann et al 1981;Kramm et al 1975;Signore et al 2012). In this respect it is notable that a113 lies close to d116Arg (His in human HbA) of the neighboring chain, conceivably forming a proton-linked Cl -binding site.…”