SOLVENT-DEPENDENT STRUCTURE AND DYNAMICS IN MYOGLOBIN

Using resonance Raman spectroscopy, we explore the effect of temperature and solvent variation on myoglobin (Mb) structure and dynamics. A 2.6-cm(-1) downshift of the iron-histidine (Fe-His) mode of deoxyMb in 75% glycerol relative to that observed in aqueous buffer indicates a glycerol-induced alteration of the heme pocket structure, possibly due to the reduced water activity in the mixed solvent. The effective photolysis yield of MbCO between 100 and 200 K is also larger in 75% glycerol samples than in frozen aqueous samples, suggesting an enhanced rate for CO recombination in the latter. Measurements of the Fe-His frequency of myoglobin in 75% glycerol as a function of time and temperature following CO photolysis allow us to directly monitor protein relaxation. The results can be described successfully using a ''glassy'' relaxation function and are not consistent with an exponential relaxation described by an Arrhenius rate law. Even at room temperature, the Fe-His frequency of the 10-ns transient MbCO photoproduct is 3 cm(-1) higher than the equilibrium deoxyMb value in 75% glycerol. In aqueous solutions, on the other hand, we confirm a previous report that the 10-ns photoproduct is spectroscopically indistinguishable from deoxyMb at room temperature. This dramatic retardation of the Fe-His relaxation in 75% glycerol is distinct from the modest solvent dependence reported for visible and near-infrared absorption bands. We propose that band III and other heme electronic transitions are sensitive to a rapid local relaxation of the heme coupled with the rearrangement of adjacent side chains, while the Fe-His frequency probes a slower global relaxation of the polypeptide backbone. Reported changes in the intensity of the Fe-His band occurring more rapidly than either of these nuclear motions may reflect the lowering of the heme electronic symmetry on subpicosecond time scales following photolysis. We discuss the properties of barrier relaxation models used to describe the effects of the evolving protein structure on recombination kinetics.