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The majority of bird species co-express two functionally distinct hemoglobin (Hb) isoforms in definitive erythrocytes: HbA (the major adult Hb isoform, with α-chain subunits encoded by the αA-globin gene) and HbD (the minor adult Hb isoform, with α-chain subunits encoded by the αD-globin gene). The αD-globin gene originated via tandem duplication of an embryonic α-like globin gene in the stem lineage of tetrapod vertebrates, which suggests the possibility that functional differentiation between the HbA and HbD isoforms may be attributable to a retained ancestral character state in HbD that harkens back to a primordial, embryonic function. To investigate this possibility and to examine other aspects of the evolution of the avian α-like globin genes, in collaborative effort with the Roy E. Weber lab, Joana Projecto-Garcia, Chandrasekhar Nataraja, and Hideaki Moriyama, we conducted a combined analysis of protein biochemistry and sequence evolution to characterize the structural and functional basis of Hb isoform differentiation in birds. The main objectives were: (1) to characterize the O2-binding properties of HbA and HbD in species that are representative of several major avian lineages; (2) to gain insight into the possible structural basis of the observed functional differentiation between the HbA and HbD isoforms; and (3) to determine whether functional differentiation between the HbA and HbD isoforms is primarily attributable to post-duplication substitutions that occurred in the αA- and αD-globin gene lineages, or whether the differentiation is attributable to substitutions that occurred in the single-copy, pre-duplication ancestor of the αD- and αE-globin genes, in which case the distinctive properties of HbD may represent a retained ancestral character state that is shared with embryonic Hb. Functional experiments involving purified HbA and HbD isofoms from 12 different bird species confirmed that HbD is characterized by a consistently higher O2-affinity in the presence of allosteric effectors such as organic phosphates and Cl- ions. In the case of both HbA and HbD, analyses of oxygenation properties under the two-state Monod-Wyman-Changeux allosteric model revealed that the pH-dependence of Hb-O2 affinity stems from changes in the O2 association constant of deoxy (T-state) Hb. Ancestral sequence reconstructions indicated that the replacement substitutions that distinguish the avian αA- and αD-globin genes occurred exclusively on post-duplication branches of the gene family phylogeny, suggesting that the observed functional differences between the HbA and HbD isoforms are not attributable to the retention of an ancestral (pre-duplication) character state in the αD-globin gene.
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