Structure and function of crocodilian hemoglobins and allosteric regulation by chloride, ATP, and CO₂

Abstract

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 CO2. 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 CO2, which would permit effective O2 unloading to tissues in response to an increase in metabolism during intense activity and diving. Although the Hbs of all crocodilians exhibit similar intrinsic O2-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 NH2-terminal acetylation. Whereas chloride appears to be a potent allosteric effector of all crocodile Hbs, ATP has a strong, chloride-independent effect on Hb-O2 affinity only in caimans. Modeling suggests that allosteric ATP binding has a somewhat different structural basis in crocodilian and mammalian Hbs.

Synthesis of Recombinant Human Hemoglobin With NH2‐Terminal Acetylation in Escherichia coli

“…7). For purified samples of each rHb, we quantified the fraction of αand β-chain peptides of NH 2 -terminal acetylation using PEAK X software (Fago et al, 2020). We also measured the oxygenation properties of native human Hb, unacetylated rHb (expressed using the pGM + pMAP system), and acetylated rHb (expressed using the pGM + pMAP + pNatA system; Fig.…”

“…This may not have major effects on the allosteric regulation of human Hb. However, in Hb of other vertebrates such as crocodilians, NH 2 -terminal residues may play a more important role in the allosteric binding of organic phosphates and other cofactors so that acetylation can produce significant functional effects (Fago et al, 2020;Weber, Fago, Malte, Storz, & Gorr, 2013). Our new protocol for synthesizing rHb with NH 2 -terminal acetylation provides a means of assessing how this post-translational modification affects the structural and functional properties of the protein.…”

New insights into the allosteric effects of CO2and bicarbonate on crocodilian hemoglobin

“…The bicarbonate sensitivity of crocodilian Hbs was first described in the spectacled caiman ( Caiman crocodilus ) (Bauer et al, 1981; Perutz et al, 1981), but the allosteric effect was inferred without direct experimental evidence of bicarbonate binding to Hb. Subsequent studies on Hbs from other crocodilian species documented a direct allosteric effect of CO 2 (Fago et al, 2020; Jensen et al, 1998; Weber et al, 2013), but the direct contribution of bicarbonate-binding has neither been confirmed in these studies nor quantified. The reason is likely that it is difficult to experimentally distinguish the allosteric effects of CO 2 from those of bicarbonate and protons because at equilibrium all these three compounds are present in solution.…”

“…Blood was immediately centrifuged at 4°C (5000 g, 5 min) to remove plasma and RBCs were then washed five times in 0.9 % NaCl, 0.5 mM EDTA and stored at −80 °C until further processing. As described previously (Fago et al, 2020), we sampled blood and prepared hemolysates from 13 other crocodilian species (American alligator, Alligator mississippiensis, [Duadin, 1802] ; Chinese alligator, A. sinensis, [Fauvel, 1879]; broad-snouted caiman, Caiman latirostris [Duadin, 1802] ; yacaré caiman, C. yacare [Duadin, 1802] ; black caiman, Melanosuchus niger, [Spix, 1825] ; smooth-fronted caiman, Paleosuchus trigonatus, [Schneider, 1801] ; American crocodile, Crocodylus acutus, [Cuvier, 1807] ; Philippine crocodile, C. mindorensis [Schmidt, 1928]; Nile crocodile, C. niloticus [Laurenti, 1768] ; New Guinea crocodile, C. novaeguineae [Schdmit, 1928] ; saltwater crocodile, C. porosus [Schneider, 1801]; Cuban crocodile, C. rhombifer [Cuvier, 1807] ; Siamese crocodile, C. siamensis [Schneider, 1801]).…”

“…The reason is likely that it is difficult to experimentally distinguish the allosteric effects of CO 2 from those of bicarbonate and protons because at equilibrium all these three compounds are present in solution. Moreover, previous studies (Bauer et al, 1981) used different experimental and buffer conditions and rarely accounted for the potential effect of other anions, such as chloride ions, which are potent allosteric modulators of crocodilian Hb-O 2 affinity (Fago et al, 2020; Storz, 2019; Weber et al, 2017).…”