518 The Journal of Experimental Biology 215, 518-525 © 2012. Published by The Company of Biologists Ltd doi:10.1242/jeb.063669 RESEARCH ARTICLE Origin and mechanism of thermal insensitivity in mole hemoglobins: a test of the ʻadditionalʼ chloride binding site hypothesis Anthony V. Signore1, Jörg Stetefeld2, Roy E. Weber3 and Kevin L. Campbell1,* 1Department of Biological Sciences, University of Manitoba, 50 Sifton Road, Winnipeg, Manitoba R3T 2N2, Canada, 2Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada and 3Zoophysiology, Department for Bioscience, University of Aarhus, DK-8000 Aarhus, Denmark *Author for correspondence ([email protected]) Accepted 5 October 2011 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 ʻadditionalʼ chloride ions within a cationic pocket between residues 8His, 76Lys and 77Asn on the -like -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 (⌬HЈ). Here we show that the variability in HЈ seen among mole Hbs cannot be attributed to differential Cl– binding at 8, 76 and 77, as the Cl– sensitivity of mole Hbs is unaffected by amino acid changes at this site (i.e. the proposed ʻadditionalʼ Cl– binding site is not operational in mole Hbs). Rather, we demonstrate that the numerically low ⌬HЈ 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 (125His and 34Glu/Asp) that are not present in other mole Hbs. Supplementary material available online at http://jeb.biologists.org/cgi/content/full/215/3/518/DC1 Key words: enthalpy, hemoglobin, molecular evolution, oxygen binding, shrew mole, star-nosed mole, Talpidae, temperature effect. INTRODUCTION given that the temperature dependence of blood–O2 affinity Mammalian hemoglobin (Hb) is composed of two -type and two (whereby Hb–O2 affinity tends to increase with decreasing -type polypeptide strands, each of which bears an iron-containing temperature) was first observed more than 100years ago (Barcroft heme group (the site of reversible O2 binding). To optimize the and King, 1909). As with blood–O2 affinity, temperature sensitivity uptake and delivery of O2, this metalloprotein switches its varies substantially among species, with the magnitude of this effect quaternary structure between high O2 affinity [predominantly resulting from a symphony of oxygenation-linked chemical oxygenated, relaxed (R)] and low O2 affinity [predominantly processes that together determine the overall enthalpy of oxygenation deoxygenated, tense (T)] states (Perutz, 1983). Although this (⌬HЈ) of the protein. Briefly, this can be formulated as: r general mechanism is perfectly conserved among mammalian Hbs, ⌬HЈ⌬HO2+⌬HH2O+⌬HT R+⌬HEffector, where ⌬HO2 is the intrinsic –1 the physicochemical properties of the protein can vary substantially heat of heme oxygenation (approximately –59kJmol O2 bound), H2O –1 TrR both within (e.g. ontogenically expressed Hb isoforms) and between ⌬H is the heat of solution (–12.55kJmol O2), ⌬H is the species (Brittain, 2002; Weber, 2007). Some of this variability has heat of the TrR transition and ⌬HEffector is the heat of effector (e.g. – + been attributed to residue replacements along the - or -type globin DPG, Cl , CO2 and H ) dissociation (Weber and Campbell, 2011). chains that perturb or strengthen the R or T state, hence modifying Because ⌬HO2 and ⌬HH2O are virtually invariant, variations in HЈ the inherent oxygenation properties of the protein (Perutz, 1983; appear to be primarily driven by changes in endothermic Weber, 2007). Alternatively, substitutions may trigger structural contributions from oxygenation-linked effector release and/or changes in the protein that alter its sensitivity to naturally occurring possibly by changes in ⌬HTrR (Weber and Campbell, 2011). heterotropic (i.e. non-oxygen) ligands – e.g. 2,3-diphosphoglycerate Notably, numerical reductions in HЈ – whereby Hb–O2 affinity is – + (DPG), chloride ions (Cl ), protons (H ) and CO2 – thereby shifting less affected by changes in temperature – may have had important the R}T equilibrium (Perutz, 1983; Weber, 2007). evolutionary consequences for a number of mammalian lineages Although heterotropic ligands are known to be important (Campbell et al., 2010a; Weber and Campbell, 2011). Indeed, a modulators of Hb–O2 affinity in vivo, the molecular mechanisms numerically low negative HЈ Hb phenotype (approximately –14 –1 underlying the effect of temperature on Hb–O2 affinity are much to –19kJmol O2) appears to be important in allowing regionally less understood (Weber and Campbell, 2011). This is surprising heterothermic (e.g. arctic and aquatic) endotherms to balance O2 THE JOURNAL OF EXPERIMENTAL BIOLOGY Thermal sensitivity of mole hemoglobins 519 delivery at the peripheral tissues in the face of temperature-induced and eastern moles) and semi-aquatic (star-nosed) moles. Hence, it changes in O2 demand at these sites (De Rosa et al., 2004; Weber was hoped that assessment of the inherent oxygenation and Campbell, 2011). characteristics and effector sensitivity of its Hb might further our We previously determined that the individual Hb components of understanding of the mechanisms underlying hypoxic adaptation in the coast mole (Scapanus orarius) and the eastern mole (Scalopus the more derived mole species. aquaticus) likewise possess numerically low enthalpies of –1 oxygenation (between –8 and –14kJmol O2) in the presence of MATERIALS AND METHODS allosteric effectors, a trait that was hypothesized to minimize the Sample collection impairment of O2 uptake at the lungs while burrowing in hypoxic Two American shrew moles, Neurotrichus gibbsii (Baird 1858), tunnels (Campbell et al., 2010b). Interestingly, the effects of were live captured in pitfall traps, one from Blaine, Whatcom temperature on the whole-blood–O2 affinity of coast, eastern and County, WA, USA, and the other from Vancouver, British star-nosed (Condylura cristata) moles indicated that the blood of Columbia, Canada. Three star-nosed moles, Condylura cristata the first two species is considerably less sensitive to temperature (Linnaeus 1758), were captured in Sherman live traps, two in than that of the latter (Campbell et al., 2010b). This finding is southeast Manitoba, Canada (Caddy Lake and Piney), and one in surprising given that the star-nosed mole is not only semi-aquatic, Potter County, PA, USA. The lack of discernable intraspecific but is distributed substantially farther north than the thermally differences in gene sequences and/or Hb–O2 binding characteristics buffered, strictly fossorial coast and eastern moles (Petersen and (see below) of individuals from these geographically separated Yates, 1980). In fact, star-nosed moles have been observed populations suggested that our findings are a good representation tunnelling in snow and even diving beneath ice during the winter for each species. Blood and tissue samples were obtained soon after (Merriam, 1884; Hamilton, 1931). Results of Campbell and anaesthesia-induced euthanization following both University of coworkers (Campbell et al., 2010b) further suggested that coast and British Columbia and University of Manitoba approved protocols eastern mole Hbs possess an extra (relative to human HbA) Cl– and with adherence to the guidelines of the Canadian Council on binding site that does not overlap with DPG binding. Like that of Animal Care. In all cases, blood samples were stored in sealed vials coast and eastern moles, bovine Hb possesses a specific and at –70°C. Fresh liver and spleen samples were excised immediately ‘additional’ (with respect to human HbA) oxygenation-linked Cl– and transferred to vials containing 95% ethanol and RNAlater binding site (De Rosa et al., 2004). Based on functional data obtained (Ambion, Austin, TX, USA), respectively, and stored similarly. from human site-directed mutants, it was proposed that this Cl– binding site resides between three cationic residues on the -globin Hb–O2 affinity chain: 8Lys, 76Lys and 77His (Fronticelli et al., 1995). Later The Hb components from two star-nosed moles (Manitoba and PA) work revealed that Hbs possessing 8Lys and 76Lys together with and a single American shrew mole (Vancouver) were isolated by His or Asn at 77 display numerically reduced ⌬HЈ values in the isoelectric focusing in a 110ml LBK sucrose density gradient presence of 0.1moll–1 Cl– relative to human HbA (–41kJmol–1); column following the procedure of Campbell et al. (Campbell et HbA possesses a neutral Ala residue at 76, which presumably al., 2010b). Measurements of the partial pressure