title: Origin and mechanism of thermal insensitivity in mole hemoglobins: a test of the 'additional' chloride binding site hypothesis.
abstract: 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 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.
authors.affiliation: Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba R3T 2N2, Canada.
authors.forename: Anthony V
authors.initials: AV
authors.lastname: Signore
authors.forename: Jörg
authors.initials: J
authors.lastname: Stetefeld
authors.forename: Roy E
authors.initials: RE
authors.lastname: Weber
authors.forename: Kevin L
authors.initials: KL
authors.lastname: Campbell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001831:Body Temperature / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism*; D006454:Hemoglobins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008971:Moles / Q000378:metabolism*; D010100:Oxygen / Q000097:blood / Q000378:metabolism*; D011522:Protons; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D005719:gamma-Globulins / Q000737:chemistry / Q000378:metabolism
chemical_list: D002712:Chlorides; D006454:Hemoglobins; D011522:Protons; D005719:gamma-Globulins; D010100:Oxygen