Crystal structure of T state haemoglobin with oxygen bound at all four haems

The cooperative binding of oxygen by haemoglobin results from restraints on ligand binding in the T state. The unfavourable interactions made by the ligands at the haems destabilise the T state and favour the high affinity R state. The T double left right arrow R equilibrium leads, in the presence of a ligand, to a rapid increase in the R state population and therefore generates cooperative binding. There is now considerable understanding of this phenomenon, but the interactions that reduce ligand affinity in the T state have not yet been fully explored, owing to the difficulties in preparing T state haemoglobin crystals in which all the subunits are oxygenated. A protocol has been developed to oxygenate deoxy T state adult human haemoglobin (HbA) crystals in air at 4 degrees C at all four haems without significant loss of crystalline order. The X-ray crystal structure, determined to 2.1 Angstrom spacing, shows significant changes in the alpha and beta haem pockets as well as changes at the alpha(1) beta(2) interface in the direction of the R quaternary structure. Most of the shifts and deviations from deoxy T state HbA are similar to, but larger than, those previously observed in the T state met and other partially liganded T state forms. They provide clear evidence of haem-haem interaction in the T state. (C) 1996 Academic Press Limited