A comparison of functional and structural consequences of the tyrosine B10 and glutamine E7 motifs in two invertebrate hemoglobins (Ascaris suum and Lucina pectinata)

Two invertebrate haemoglobins (Hb) from Ascaris suum and Lucina pectinata in which residues E7 and B10 are a glutamine and tyrosine, respectively, and which have high oxygen affinities stemming from slow ligand off rates were compared with respect to conformational and functional properties. Ultraviolet and visible resonance Raman spectroscopy was used to probe the ligand-dependent hydrogen bonding pattern of the tyrosine residues and the proximal haem pocket interactions. Fourier transform infrared absorption spectroscopy was used to probe the dielectric properties of the distal haem pocket through the stretching frequency of CO bound to the haem. Functionality was probed through the geminate rebinding of both CO and O2. The findings revealed 2 very different patterns indicating 2 different mechanisms for achieving low oxygen off rates. In Ascaris Hb, a hydrogen bonding network that included the E7 Gln, B10 Try and O2 bound to the haem resulted in a tight cage for the O2. Dissociation of the O2 required a large amplitude conformational fluctuation that resulted both in spontaneous dissociation of the O2 through the loss of hydrogen bond stabilization and an enhanced probability for ligand escape though the transient disruption and opening of the cage. In the case of Lucina Hb, there was no evidence for a tight cage and the data support a model in which the hydrogen bonding network is more tenuous and the equilibrium state of distal pocket is more open and accessible than in Ascaris. The results explain why Ascaris Hb has one of the highest oxygen affinities known (P50 ~ 10-3 Torr) while Lucina Hb II has an oxygen affinity comparable to that of Mb (P50 = 0.13 Torr), even though both of these Hbs contain the B10 Tyr and E7 Gln motif and display very low oxygen off rates..