Temperature-jump studies and polarized absorption spectroscopy of methemoglobin-thiocyanate single crystals

Association equilibria and association kinetics of the thiocyanate binding reaction to methemoglobin in single crystals and solution are studied using temperature-jump technique and polarized absorption spectroscopy. Different kinetic constants are found for the reaction in solution and crystal phase for the α- and β-subunits of the methemoglobin tetramer. The reduction of the reactivity of the α- and β-subunits in crytsalline phase is 6-fold and 2.4-fold, respectively, compared to the values found in solution. The intramolecular binding reaction of the N_ε of the distal histidine E7 which is observed in methemoglobin in solution cannot be detected in single crystals. Our results suggest that crystallization of hemoglobin has little influence on small-scale structural fluctuations which are necessary for ligands to get to the binding sites and large-scale structural motions are suppressed.