SOLVENT-DEPENDENT STRUCTURE AND DYNAMICS IN MYOGLOBIN

Using resonance Raman spectroscopy, we explore the effect of temperatu re 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 g lycerol-induced alteration of the heme pocket structure, possibly due to the reduced water activity in the mixed solvent. The effective phot olysis yield of MbCO between 100 and 200 K is also larger in 75% glyce rol samples than in frozen aqueous samples, suggesting an enhanced rat e for CO recombination in the latter. Measurements of the Fe-His frequ ency of myoglobin in 75% glycerol as a function of time and temperatur e following CO photolysis allow us to directly monitor protein relaxat ion. The results can be described successfully using a ''glassy'' rela xation function and are not consistent with an exponential relaxation described by an Arrhenius rate law. Even at room temperature, the Fe-H is frequency of the 10-ns transient MbCO photoproduct is 3 cm(-1) high er than the equilibrium deoxyMb value in 75% glycerol. In aqueous solu tions, on the other hand, we confirm a previous report that the 10-ns photoproduct is spectroscopically indistinguishable from deoxyMb at ro om temperature. This dramatic retardation of the Fe-His relaxation in 75% glycerol is distinct from the modest solvent dependence reported f or visible and near-infrared absorption bands. We propose that band II I and other heme electronic transitions are sensitive to a rapid local relaxation of the heme coupled with the rearrangement of adjacent sid e 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 motio ns may reflect the lowering of the heme electronic symmetry on subpico second 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.