The Structural and Functional Analysis of the Hemoglobin D Component from Chicken

Abstract

Oxygen binding by chicken blood shows enhanced cooperativity at high levels of oxygen saturation. This implies that deoxy hemoglobin tetramers self-associate. The crystal structure of an R-state form of chicken hemoglobin D has been solved to 2.3-Å resolution using molecular replacement phases derived from human oxyhemoglobin. The model consists of an ␣ 2 ␤ 2 tetramer in the asymmetric unit and has been refined to a R-factor of 0.222 (R-free ‫؍‬ 0.257) for 29,702 reflections between 10.0-and 2.3-Å resolution. Chicken Hb D differs most from human oxyhemoglobin in the AB and GH corners of the ␣ subunits and the EF corner of the ␤ subunits. Reanalysis of published oxygen binding data for chicken Hbs shows that both chicken Hb A and Hb D possess enhanced cooperativity in vitro when inositol hexaphosphate is present. The electrostatic surface potential for a calculated model of chicken deoxy-Hb D tetramers shows a pronounced hydrophobic patch that involves parts of the D and E helices of the ␤ subunits. This hydrophobic patch is a promising candidate for a tetramer-tetramer interface that could regulate oxygen binding via the distal histidine.The oxygen binding curves of chicken blood samples taken at different stages of development exhibit Hill coefficients which exceed 4.0 (super-cooperativity) at oxygen saturation levels between 70 and 90% oxygenation (1-5). Elevated levels of oxygen-binding cooperativity should be advantageous because such hemoglobins (Hbs) 1 would deliver a greater quantity of oxygen to the tissues under normal physiological conditions. Super-cooperativity has also been observed in the blood and Hbs of several species of amphibians (6 -8), reptiles (9 -10), other birds (1, 11), and embryonic mammals (12-14). Although incomplete oxygen saturation resulted in spuriously high Hill coefficients in much of the early data (see , the data of Lapennas and Reeves (5) on chicken blood and Holland et al.(12-14) on embryonic marsupial blood were obtained at saturating pressures of O 2 .The blood of adult chickens, as in most other birds, is composed of two Hb components that have identical ␤ chains but differ in the sequences of the ␣ subunits. Hb A (␣ The cooperative binding of oxygen by tetrameric Hbs arises from the transition of the Hb from a conformation with a low oxygen affinity (T-state) to a conformation with a high oxygen affinity (R-state) (21,22). Much of our understanding of this process has come from the crystallographic analysis of the unligated (T-state) and ligated (R-state) structures of both human Hb A (23-26) and horse . Although a structural explanation of super-cooperativity requires the crystallographic analysis of both the R-and T-state forms of chicken Hb D, the analysis of intermediate structures are likely to be necessary for understanding the mechanism of cooperativity because knowledge of two thermodynamic states does not itself determine the pathway between them. We report here the structure of an R-state form of chicken Hb D solved with diffraction data to 2.3-Å resolution. Th...

Structural and functional analysis of the two haemoglobins of the Antarctic seabird Catharacta maccormicki

“…In fact, the two Hbs have the b chain in common. Moreover, the models reported here are very similar to those suggested by Zhang et al [3] and Knapp et al [24] in goose and chicken Hbs, respectively.…”

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

“…Our sequence data have implications for long standing questions about the specific residue(s) that mediate the intertetramer interactions between deoxy-HbDs (22)(23)(24). Because avian HbA and HbD share a common ␤-chain, interactions between deoxy-HbD tetramers must be mediated by surface residues on the ␣ D -chain subunit that are not shared with the ␣ A -chain.…”

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Structural studies of haemoglobin from pisces species shortfin mako shark (Isurus oxyrinchus)at 1.9 Å resolution

“…for 1 h, which yielded cell-free Hb solution as the supernatant. The sample was applied onto an ion exchange chromatography column using DEAE-cellulose as the column material; the chromatographic column was equilibrated with water (Knapp et al, 1999). The salt-gradient elution was achieved using a NaCl gradient from 0.1 to 1.0 M. The Hb was eluted at 0.1 M NaCl and was collected at a rate of 3 ml min À1 .…”