Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

Abstract

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3(-)), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3(-) levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3(-) binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues.

Keywords: Bohr effect; allosteric interaction; carbon dioxide; crocodilians; oxygen-binding.

Fig. 1.

Amino acid sequences of the α and β globins of dwarf caiman (Paleosuchus palpebrosus) hemoglobin (Hb) compared with those for spectacled caiman (Caiman crocodilus), Nile crocodile (Crocodylus niloticus), the American alligator (Alligator mississippiensis) (51), chicken, and human. Residues in the other species are shown only where they differ from those in human Hbs. His residues highlighted in yellow and blue are titratable (surface) residues and nontitratable residues, respectively, in human Hb (11, 53); those highlighted in green represent potential gains of titratable His residues compared with human Hb.

Fig. 2.

Oxygen tension (P₅₀) and Hill's cooperativity coefficients (n₅₀) at half O₂ saturation of P. palpebrosus Hb measured at 10°C (triangles and diamonds) and 25°C (full and half circles), in 0.05 M HEPES buffer (circles and diamonds) or 0.05 M MES buffer (half-circles and triangles) and in the absence (open symbols) and presence (closed symbols) of 0.1 M Cl⁻. Heme concentration, 0.37 mM.

Fig. 3.

A: P₅₀ and n₅₀ values of P. palpebrosus Hb measured in 50 mM HEPES buffer at 10° and 25°C in the absence (−) and presence (+) of 0.1 M Cl⁻, and the absence (open symbols) and presence (closed symbols) of saturating levels [ATP/Hb and 2,3-diphosphoglycerate (DPG)/Hb ratio 6.2; inositol hexaphosphate (IHP)/Hb ratio ∼28] of the polyanionic phosphate effectors (Pⁿ⁻) ATP, DPG, and IHP. B: histograms showing the P₅₀ values of the stripped Hb (str., open columns) and the log P₅₀ shifts (shaded columns) induced by chloride, ATP, DPG, and IHP at pH 7.4 and 10° and 25° C. Other conditions as in Fig. 2.

Fig. 4.

P₅₀ and n₅₀ of P. palpebrosus Hb at pH 7.4 and 25°C in the absence (open symbols) and presence (solid symbols) of 0.1 M Cl⁻; the absence of organic phosphates (stars); and the presence of ATP (circles), DPG (triangles), and IHP (squares) at different phosphate/tetrameric Hb ratios, measured in 0.05 M HEPES buffer. Fine dotted line shows a slope of 0.25 expected if deoxygenation of the Hb molecules were linked to binding of one phosphate molecule. Heme concentration, 0.36 mM. (This figure replots earlier published P₅₀ values (96) against the [free phosphate]/[Hb] ratios).

Fig. 5.

A: pH dependence of P₅₀ and n₅₀ values of P. palpebrosus Hb in the presence of 0, 1, and 4% CO₂ (Pco₂ = 0, 7.4, and 29.4 mm, respectively), measured at 25°C in 50 mM HEPES buffer. B: the specific (pH-independent) effect of CO₂ on Hb-O₂ affinity at pH 7.0 and 7.4. Heme concentration 0.37 mM.

Fig. 6.

Extended Hill plots of P. palpebrosus Hb at pH ∼7.60 (half circles) and ∼6.96 (triangles) in the absence (open symbols) and presence (closed symbols) of 0.10 M Cl⁻. Heme concentration, 0.37 mM.

Fig. 7.

Elution profile of oxygenated (○) and deoxygenated (●) P. palpebrosus Hb on a column Sephacryl S-200 HR gel showing fractions pooled for analyses (open horizontal bars 1–4). Inset: K_av values of these fractions compared with those of human Hb A and proteins of known molecular mass, viz., cyt.C (cytochrome c), Mb (myoglobin), oval albumin (O.A.), bovine serum albumin (B.S.A.), aldolase (Ald.), catalase (Cat.), and ferritin (Ferr.).

Fig. 8.

P₅₀ values and pH dependence of three molecular mass fractions (fr.) of P. palpebrosus Hb isolated by gel filtration (cf. Fig. 7), measured at 25°C in HEPES (▵, ○, and ▫) and MES (◇) buffers.