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Abstract: This study examines the functional and structural effects of amino acid substitution at α1β2 interface of Hb Santa Clara (β97His→Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding 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 β97 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 βFG4 position has been further examined by computational models of known βFG4 variants, namely Hb Malmö (β97His→Gln), Hb Wood (β97His→Leu), Hb Nagoya (β97His→Pro) and Hb Moriguchi (β97His→Tyr). These findings demonstrate that, among the various residues at the α1β2 (and α2β1) intersubunit interface, His βFG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin. [Copyright &y& Elsevier]