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

   https://doi.org/https://doi.org/10.1073/pnas.2023936118  

   Signore, Anthony V; Tift, Michael S; Hoffman, Federico G; Schmitt, Todd L; Moriyama, Hideaki; Storz, Jay F. (March 2021, Proceedings of the National Academy of Sciences of the United States of America)

   Somero, George N. (Ed.)

   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. 

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2. Adrenergically induced translocation of red blood cell β-adrenergic sodium-proton exchangers has ecological relevance for hypoxic and hypercapnic white seabass

   https://doi.org/10.1152/ajpregu.00175.2021  

   Harter, Till S.; Clifford, Alexander M.; Tresguerres, Martin (November 2021, American Journal of Physiology-Regulatory, Integrative and Comparative Physiology)

   White seabass ( Atractoscion nobilis) increasingly experience periods of low oxygen (O 2 ; hypoxia) and high carbon dioxide (CO 2 , hypercapnia) due to climate change and eutrophication of the coastal waters of California. Hemoglobin (Hb) is the principal O 2 carrier in the blood and in many teleost fishes Hb-O 2 binding is compromised at low pH; however, the red blood cells (RBC) of some species regulate intracellular pH with adrenergically stimulated sodium-proton-exchangers (β-NHEs). We hypothesized that RBC β-NHEs in white seabass are an important mechanism that can protect the blood O 2 -carrying capacity during hypoxia and hypercapnia. We determined the O 2 -binding characteristics of white seabass blood, the cellular and subcellular response of RBCs to adrenergic stimulation, and quantified the protective effect of β-NHE activity on Hb-O 2 saturation. White seabass had typical teleost Hb characteristics, with a moderate O 2 affinity (Po 2 at half-saturation; P 50 2.9 kPa) that was highly pH-sensitive (Bohr coefficient −0.92; Root effect 52%). Novel findings from super-resolution microscopy revealed β-NHE protein in vesicle-like structures and its translocation into the membrane after adrenergic stimulation. Microscopy data were corroborated by molecular and phylogenetic results and a functional characterization of β-NHE activity. The activation of RBC β-NHEs increased Hb-O 2 saturation by ∼8% in normoxic hypercapnia and by up to ∼20% in hypoxic normocapnia. Our results provide novel insight into the cellular mechanism of adrenergic RBC stimulation within an ecologically relevant context. β-NHE activity in white seabass has great potential to protect arterial O 2 transport during hypoxia and hypercapnia but is less effective during combinations of these stressors. 

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3. Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO ₂

   https://doi.org/10.1152/ajpregu.00342.2019  

   Fago, Angela; Natarajan, Chandrasekhar; Pettinati, Martín; Hoffmann, Federico G.; Wang, Tobias; Weber, Roy E.; Drusin, Salvador I.; Issoglio, Federico; Martí, Marcelo A.; Estrin, Darío; et al (March 2020, American Journal of Physiology-Regulatory, Integrative and Comparative Physiology)

   Hemoglobins (Hbs) of crocodilians are reportedly characterized by unique mechanisms of allosteric regulatory control, but there are conflicting reports regarding the importance of different effectors, such as chloride ions, organic phosphates, and CO 2 . Progress in understanding the unusual properties of crocodilian Hbs has also been hindered by a dearth of structural information. Here, we present the first comparative analysis of blood properties and Hb structure and function in a phylogenetically diverse set of crocodilian species. We examine mechanisms of allosteric regulation in the Hbs of 13 crocodilian species belonging to the families Crocodylidae and Alligatoridae. We also report new amino acid sequences for the α- and β-globins of these taxa, which, in combination with structural analyses, provide insights into molecular mechanisms of allosteric regulation. All crocodilian Hbs exhibited a remarkably strong sensitivity to CO 2 , which would permit effective O 2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O 2 -affinities, there is considerable variation in sensitivity to Cl − ions and ATP, which appears to be at least partly attributable to variation in the extent of NH 2 -terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O 2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs. 

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4. Adaptive Changes in Hemoglobin Function in High-Altitude Tibetan Canids Were Derived via Gene Conversion and Introgression

   https://doi.org/10.1093/molbev/msz097  

   Signore, Anthony V; Yang, Ying-Zhong; Yang, Quan-Yu; Qin, Ga; Moriyama, Hideaki; Ge, Ri-Li; Storz, Jay F; Wilke, Claus (July 2019, Molecular Biology and Evolution)

   Abstract A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb–O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the β-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked β-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the β-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb–O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed’s relatively short duration of residency at high altitude. 

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5. New insights into the allosteric effects of CO2 and bicarbonate on crocodilian hemoglobin

   https://doi.org/10.1242/jeb.242615  

   Bautista, Naim M.; Malte, Hans; Natarajan, Chandrasekhar; Wang, Tobias; Storz, Jay F.; Fago, Angela (August 2021, Journal of Experimental Biology)

   ABSTRACT Crocodilians are unique among vertebrates in that their hemoglobin (Hb) O2 binding is allosterically regulated by bicarbonate, which forms in red blood cells upon hydration of CO2. Although known for decades, this remarkable mode of allosteric control has not yet been experimentally verified with direct evidence of bicarbonate binding to crocodilian Hb, probably because of confounding CO2-mediated effects. Here, we provide the first quantitative analysis of the separate allosteric effects of CO2 and bicarbonate on purified Hb of the spectacled caiman (Caiman crocodilus). Using thin-layer gas diffusion chamber and Tucker chamber techniques, we demonstrate that both CO2 and bicarbonate bind to Hb with high affinity and strongly decrease O2 saturation of Hb. We propose that both effectors bind to an unidentified positively charged site containing a reactive amino group in the low-O2 affinity T conformation of Hb. These results provide the first experimental evidence that bicarbonate binds directly to crocodilian Hb and promotes O2 delivery independently of CO2. Using the gas diffusion chamber, we observed similar effects in Hbs of a phylogenetically diverse set of other caiman, alligator and crocodile species, suggesting that the unique mode of allosteric regulation by CO2 and bicarbonate evolved >80–100 million years ago in the common ancestor of crocodilians. Our results show a tight and unusual linkage between O2 and CO2 transport in the blood of crocodilians, where the build-up of erytrocytic CO2 and bicarbonate ions during breath-hold diving or digestion facilitates O2 delivery, while Hb desaturation facilitates CO2 transport as protein-bound CO2 and bicarbonate. 

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