Heme-solvent coupling: a Mössbauer study of myoglobin in sucrose.

The Mössbauer effect of 57Fe-enriched samples was used to investigate the coupling of 80% sucrose/water, a protein-stabilizing solvent, to vibrational and diffusive modes of the heme iron of CO-myoglobin. For comparison we also determined the Mössbauer spectra of K4 57Fe (CN)6 (potassium ferrocyanide, PFC), where the iron is fully exposed in the same solvent. The temperature dependence of the Mössbauer parameters derived for the two samples proved to be remarkably similar, indicative of a strong coupling of the main heme displacements to the viscoelastic relaxation of the solvent. We show that CO escape out of the heme pocket couples to the same type of fluctuations, whereas intramolecular bond formation involves solvent-decoupled heme deformation modes that are less prominent in the Mössbauer spectrum. With respect to other solvents, however, sucrose shows a reduced viscosity effect on heme displacements and the kinetics of ligand binding due to preferential hydration of the protein. This result confirms thermodynamic predictions of the stabilizing action of sucrose by a dynamic method.

Solvent composition and viscosity effects on the kinetics of CO binding to horse myoglobin.

Ligand binding to myoglobin in aqueous solution involves two kinetic components, one extramolecular and one intramolecular, which have been interpreted in terms of two sequential kinetic barriers. In mixed solvents and sub-zero temperatures, the outer barrier increases and the inner barrier splits into several components, giving rise to fast intramolecular recombination. The nature of these barriers and their relation to structural relaxation are examined using the effect of solvent composition and viscosity on the kinetics of CO binding to horse myoglobin in 60% ethylene glycol/water, 75% and 90% glycerol/water, 80% and 92% sucrose/water solutions. Measurements of the corresponding solvent structural relaxation rates by frequency resolved calorimetry allow us to discriminate between solvent composition and viscosity-related effects. The outer kinetic barrier controlling ligand entry and release depends on the viscosity consistent with Kramers-Stokes law of activated escape in the presence of friction. At high cosolvent concentration, we observe deviations from Stokes law, implying a smaller microviscosity at the protein-solvent interface as compared to the bulk. The inner barrier and its coupling to structural relaxation appears to be independent of viscosity but changes with solvent composition. As a possible explanation, we discuss the role of distal water molecules in the formation of the effective inner barrier. At low temperatures, this barrier has a distributed height, depending only slightly on the nature of the cosolvent and temperature at low cosolvent concentrations. In contrast, myoglobin embedded in a sucrose glass (92% sucrose/water) exhibits a temperature-dependent and bimodal enthalpy distribution. This result demonstrates that the exchange between protonation states of His64, A0 left and right arrow A1, can take place in the glass and at temperatures as low as 80 K.