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

Abstract

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1a, G6a and HC3a, is located between the two a chains. This additional site may act as an entry/ leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two b chains by means of a site±site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.Keywords: Antarctica; Bohr effect; haemoglobin; homology modelling; phosphate binding.Antarctic organisms are exposed to very low temperatures. Thus, in order to face extreme life conditions, suitable mechanisms of cold adaptation have been developed, involving physiological and biochemical processes.In the framework of a study on structure±function relationships in haemoglobin (Hb) and myoglobin (Mb), we began to investigate possible adaptations for oxygen transport and release in Antarctic birds. Studies on the respiratory proteins of the Emperor penguin (Aptenodytes forsteri ) have also been reported [1,2]. These studies showed that special functional features have been acquired in order to meet special physiological needs. In particular, the shape of the Bohr effect curve of Hb, as well as the very high Mb oxygen affinity, in keeping with the need of muscle to store oxygen required during prolonged exercise, appear well adapted for gas exchange during dives.Herewith we report the amino-acid sequence and the oxygen binding properties of the two Hbs of the south polar skua (Catharacta maccormicki, Stercoraridae), a seabird that breeds in coastal Antarctic regions and which has behavioural and physiological characteristics which differ from those of penguins. The two Hbs showed different heats of oxygenation in response to the physiological effectors and a weak alkaline Bohr effect which, in contrast with the hypothesis of Zhang et al. [3], is present also in the absence of chloride ions. Moreover, our experimental evidence suggests that the two Hbs contain an additional phosphate binding site. Previous studies on other Hbs [4,5] had also proposed an additional site, and suggested that i...

Gene Duplication and the Evolution of Hemoglobin Isoform Differentiation in Birds

“…The molecular dynamics simulations also predicted that the "additional" ␣-chain phosphate-binding site of HbD (sensu Tamburrini et al (18) and Riccio et al (53)) has a slightly lower IHP-binding energy relative to that of HbA (Table 4) and that the bound IHP molecule is lodged more deeply in the ␣-chain binding cleft of HbD (Fig. 6).…”

“…This indicates that HbD is less responsive to the inhibitory effects of IHP, a potent allosteric effector that preferentially binds and stabilizes the low affinity T-state quaternary structure of the Hb tetramer. The uniform difference in IHP sensitivity between HbA and HbD is surprising because the main polyphosphate-binding site is formed by a cluster of positively charged ␤-chain residues that line the interior of the central cavity (18,84). Because HbA and HbD share identical ␤-chain subunits (and thus share the same phosphatebinding sites), the observed isoform differences in IHP sensitivity must be attributable to one or more substitutions between ␣ A -and ␣ D -globin that do not directly affect the main phosphate-binding site.…”

“…Because HbA and HbD share identical ␤-chain subunits (and thus share the same phosphatebinding sites), the observed isoform differences in IHP sensitivity must be attributable to one or more substitutions between ␣ A -and ␣ D -globin that do not directly affect the main phosphate-binding site. Experimental evidence suggests that an additional polyphosphate-binding site is formed by seven residues from each ␣-chain (sites 1, 95, 99, 134, 137, 138, and 141), which stabilize IHP via charge-charge interactions (18,85,86). Specifically, ␣Lys-99 and charged residues at the ␣-chain N and C termini of avian HbA and HbD are predicted to form six salt bridges with the negatively charged phosphate groups of IHP (18,53).…”

“…Experimental evidence suggests that an additional polyphosphate-binding site is formed by seven residues from each ␣-chain (sites 1, 95, 99, 134, 137, 138, and 141), which stabilize IHP via charge-charge interactions (18,85,86). Specifically, ␣Lys-99 and charged residues at the ␣-chain N and C termini of avian HbA and HbD are predicted to form six salt bridges with the negatively charged phosphate groups of IHP (18,53). This additional phosphate-binding site is hypothesized to serve as an "entry/leaving site," which modulates Hb-O 2 affinity by enhancing phosphate uptake and transfer to the main oxygenation-linked binding site between the ␤-chain subunits (18,53).…”

“…Specifically, ␣Lys-99 and charged residues at the ␣-chain N and C termini of avian HbA and HbD are predicted to form six salt bridges with the negatively charged phosphate groups of IHP (18,53). This additional phosphate-binding site is hypothesized to serve as an "entry/leaving site," which modulates Hb-O 2 affinity by enhancing phosphate uptake and transfer to the main oxygenation-linked binding site between the ␤-chain subunits (18,53). Of the seven ␣-chain residues that compose this additional phosphate-binding site, four represent CBD sites that distinguish avian ␣ A -and ␣ D -globin sequences (1, 134, 137, and 138).…”