| Summary: | As the inherent affinity of hemoglobin (Hb) for O2 is inversely related to temperature, regionally heterothermic mammals are expected to experience reduced O2 offloading to cool, poorly insulated appendages. Presumably to compensate, select species have evolved Hb proteins with reduced thermal sensitivity that can maintain adequate O2 delivery at low temperatures. This phenotype has primarily been attributed to the binding of additional allosteric effectors to the Hb moiety relative to those of non-cold adapted species. However, we find that Hb from the extinct Steller’s sea cow binds fewer allosteric effectors than those of its tropical relatives (dugongs and manatees), yet surprisingly, the presence of these ligands reduces its thermal sensitivity to a greater extent. In an attempt to clarify the role of heterotropic ligands in the thermal sensitivity of Hb, we measured the O2 affinity of six paenungulate Hbs (woolly mammoth, Asian elephant, Steller’s sea cow, dugong, ancestral dugongid, and Florida manatee) in the presence of serially increasing Cl- and 2,3-diphosphoglycerate (DPG) concentrations at both 25 and 37C. Quantitation of effector binding revealed that the number of oxygenation-linked effector molecules bound to the Hb moiety is unrelated to the thermal sensitivity of Hb. Instead, our results suggest the thermodynamic properties of discrete Cl- and DPG sites are variable.We propose these differences are the result of epistatic interactions, in which amino acid differences in close vicinity to these sites alter the enthalpy change of effector binding. Similar epistatic interactions appear to be present in the Hbs of other mammals and have likely played a crucial role in the exploitation of cold environments.
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