Solvent modulation of the structural heterogeneity in FeIII myoglobin samples: a low temperature EPR investigation.

High spin FeIII myoglobin samples in solutions with different solvent composition have been investigated at low temperature by Electron Paramagnetic Resonance spectroscopy. The g = 6 line of the spectrum has been analyzed in terms of a distribution of the two crystal field parameters delta 1 and delta 2. By means of the Angular Overlap Method, it has been shown that these distributions entail, in turn, a distribution in the iron-heme displacement along the normal to the heme-plane. The spread in this iron-heme distance, which can be connected with the binding action of the proximal histidine, has been proposed as a quantitative measurement of the structural heterogeneity (conformational substate landscape) displayed by the protein molecules. The results point out, moreover, that the solvent composition can affect the structural heterogeneity of the protein system. In particular, addition of glycerol, ethylene glycol and sucrose yields a significant reduction in the spread of the iron-heme displacement, while the presence of ammonium sulfate induces a change in the average position of the iron in the heme-plane. The role played by the solvent in the structure and dynamics of the protein, in connection also with the conformational substate distribution, is discussed.