Adaptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. Here we investigate these possibilities by conducting a combined evolutionary and functional analysis of duplicated globin genes in natural populations of deer mice (Peromyscus maniculatus) that are adapted to different elevational zones. A multilocus analysis of nucleotide polymorphism and linkage disequilibrium revealed that high-altitude adaptation of deer mouse hemoglobin involves parallel functional differentiation at multiple unlinked gene duplicates: two ␣-globin paralogs on chromosome 8 and two ␤-globin paralogs on chromosome 1. Differences in O 2-binding affinity of the alternative ␤-chain hemoglobin isoforms were entirely attributable to allelic differences in sensitivity to 2,3-diphosphoglycerate (DPG), an allosteric cofactor that stabilizes the low-affinity, deoxygenated conformation of the hemoglobin tetramer. The two-locus ␤-globin haplotype that predominates at high altitude is associated with suppressed DPGsensitivity (and hence, increased hemoglobin-O 2 affinity), which enhances pulmonary O2 loading under hypoxia. The discovery that allelic differences in DPG-sensitivity contribute to adaptive variation in hemoglobin-O 2 affinity illustrates the value of integrating evolutionary analyses of sequence variation with mechanistic appraisals of protein function. Investigation into the functional significance of the deer mouse ␤-globin polymorphism was motivated by the results of population genetic analyses which revealed evidence for a history of divergent selection between elevational zones. The experimental measures of O 2-binding properties corroborated the tests of selection by demonstrating a functional difference between the products of alternative alleles.A daptive modifications of heteromeric proteins may involve genetically based changes in single subunit polypeptides or parallel changes in multiple genes that encode distinct, interacting subunits. The tetrameric hemoglobin (Hb) protein, which transports O 2 from the respiratory surfaces to metabolizing tissues, is an ideal molecule for addressing questions about the functional evolution of allosteric, multimeric proteins. In jawed vertebrates, Hb is a heterotetramer, consisting of two ␣-chain subunits and two ␤-chain subunits that form two semirigid ␣␤ dimers (␣ 1 ␤ 1 and ␣ 2 ␤ 2 ). A mutual rotation of the two ␣␤ dimers occurs during the oxygenation-linked transition in quaternary structure between the deoxy-and oxyHb conformations that is basic to cooperativity (1). Because modifications of Hb function are often implicated in adaptation to environmental hypoxia, and because much is known about structure-function relationships of vertebrate Hbs and their role in blood-O 2 transport (2, 3), the study of Hb function in vertebrate species that are native to hypoxic environments provides an opportunity to elucidate detailed molecular mechanisms of p...

SUMMARY In high-altitude vertebrates, adaptive changes in blood–O2 affinity may be mediated by modifications of hemoglobin (Hb) structure that affect intrinsic O2 affinity and/or responsiveness to allosteric effectors that modulate Hb–O2 affinity. This mode of genotypic specialization is considered typical of mammalian species that are high-altitude natives. Here we investigated genetically based differences in Hb–O2 affinity between highland and lowland populations of the deer mouse (Peromyscus maniculatus), a generalist species that has the broadest altitudinal distribution of any North American mammal. The results of a combined genetic and proteomic analysis revealed that deer mice harbor a high level of Hb isoform diversity that is attributable to allelic polymorphism at two tandemly duplicated α-globin genes and two tandemly duplicated β-globin genes. This high level of isoHb diversity translates into a correspondingly high level of interindividual variation in Hb functional properties. O2 equilibrium experiments revealed that the Hbs of highland mice exhibit slightly higher intrinsic O2 affinities and significantly lower Cl– sensitivities relative to the Hbs of lowland mice. The experiments also revealed distinct biochemical properties of deer mouse Hb related to the anion-dependent allosteric regulation of O2 affinity. In conjunction with previous findings, our results demonstrate that modifications of Hb structure that alter allosteric anion sensitivity play an important role in the adaptive fine-tuning of blood–O2 affinity.

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