Evolved increases in hemoglobin-oxygen affinity and the Bohr effect coincided with the aquatic specialization of penguins

Abstract

Dive capacities of air-breathing vertebrates are dictated by onboard O₂ stores, suggesting that physiologic specialization of diving birds such as penguins may have involved adaptive changes in convective O₂ transport. It has been hypothesized that increased hemoglobin (Hb)-O₂ affinity improves pulmonary O₂ extraction and enhances the capacity for breath-hold diving. To investigate evolved changes in Hb function associated with the aquatic specialization of penguins, we integrated comparative measurements of whole-blood and purified native Hb with protein engineering experiments based on site-directed mutagenesis. We reconstructed and resurrected ancestral Hb representing the common ancestor of penguins and the more ancient ancestor shared by penguins and their closest nondiving relatives (order Procellariiformes, which includes albatrosses, shearwaters, petrels, and storm petrels). These two ancestors bracket the phylogenetic interval in which penguin-specific changes in Hb function would have evolved. The experiments revealed that penguins evolved a derived increase in Hb-O₂ affinity and a greatly augmented Bohr effect (i.e., reduced Hb-O₂ affinity at low pH). Although an increased Hb-O₂ affinity reduces the gradient for O₂ diffusion from systemic capillaries to metabolizing cells, this can be compensated by a concomitant enhancement of the Bohr effect, thereby promoting O₂ unloading in acidified tissues. We suggest that the evolved increase in Hb-O₂ affinity in combination with the augmented Bohr effect maximizes both O₂ extraction from the lungs and O₂ unloading from the blood, allowing penguins to fully utilize their onboard O₂ stores and maximize underwater foraging time.

Keywords: Bohr effect; adaptation; hemoglobin; hypoxia; penguins.

Fig. 1.

Diagrammatic phylogeny showing the relationship among Sphenisciformes (penguins), Procellariiformes, and Pelecaniformes. Ancestral Hbs were reconstructed for the two indicated nodes: AncSphen and AncPro (the super order that contains Sphenisciformes and Procellariiformes). Divergence times are adapted from Claramunt and Cracraft (56).

Fig. 2.

P₅₀ values for penguin whole-blood and purified Hb at 37 °C, in the absence (stripped) and presence of 100 mM KCl and 0.2 mM IHP (+KCl +IHP). The higher the P₅₀, the lower the Hb-O₂ affinity. Whole-blood P₅₀ values are presented as mean ± SE (n = 3). Purified Hb P₅₀ values are derived from plots of logP₅₀ vs. pH in which a linear regression was fit to estimate P₅₀ at exactly pH 7.40 (± SE of the regression estimate).

Fig. 3.

Structural (A–C) and physiological (D and E) effects of amino acid substitutions in the reconstructed Hb proteins of the penguin ancestor (AncSphen) and the last common ancestor penguins shared with Procellariiformes (AncPro). (A) Molecular model of the AncSphen Hb tetramer, with the black box indicating the regions highlighted in B and C. (B) Molecular model of AncSphen Hb showing intersubunit stabilizing H bonds (pink) between β119Ser and both α111Ile and β120Lys. (C) Molecular model of AncPro Hb showing that replacement of β119Ser with Thr removes the intersubunit stabilizing H bonds. (D) Hb-O₂ affinity (as measured by P₅₀) of AncSphen, AncPro, and two mutant rHbs with penguin-specific amino acid replacements introduced on the AncPro background: AncProβ119Ser and AncPro+4. See the text for an explanation of the choice of candidate sites for mutagenesis experiments. Measurements were performed on Hb solutions (0.1 mM Hb in 0.1 M Hepes/0.5 mM EDTA) at 37 °C in the absence (stripped) and presence of +KCl +IHP. P₅₀ values are derived from plots of logP₅₀ vs. pH, in which a linear regression was fit to estimate P₅₀ at exactly pH 7.40 (± SE of the regression estimate). (E) Bohr coefficients (Δlog P₅₀/ΔpH) were estimated from plots of logP₅₀ vs. pH in which the Bohr effect is represented by the slope of the linear regression (± SE of the slope estimate).