Hb Santa Clara (beta 97His -> Asn), a human haemoglobin variant: Functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha1beta2 interface of Hb Santa Clara (beta97His->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygenbinding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of betaFG4 position has been further examined by computational models of known betaFG4 variants, namely Hb Malmö (beta97His->Gln), Hb Wood (beta97His->Leu), Hb Nagoya (beta97His->Pro) and Hb Moriguchi (beta97His->Tyr). These findings demonstrate that, among the various residues at the alpha1beta2 (and alpha2beta1) intersubunit interface, His ?FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.