GEMINATE REBINDING AND CONFORMATIONAL DYNAMICS OF MYOGLOBIN EMBEDDED IN A GLASS AT ROOM-TEMPERATURE

Below the glycerol/water glass transition (similar to 180 K), myoglobi n exhibits distributed geminate rebinding kinetics as a result of ''fr ozen'' conformational substates (Austin et al. Biochemistry 1975, 14, 5355). As the temperature is increased through the solvent glass trans ition, the apparent rate of geminate rebinding decreases. This slowing has been attributed to a protein relaxation that impedes CO rebinding at high T, but that is itself prevented at low T by energetic barrier s to conformational change (Steinbach et al. Biochemistry 1991, 30, 39 55). Using time-resolved spectroscopy with nanosecond lasers, we have studied ligand rebinding in sperm whale MbCO embedded in a glass at ro om temperature, Over a wide temperature range T = 105-297 K, the kinet ics of rebinding are well characterized by the same inhomogeneous dist ribution g(H-BA) of enthalpy barriers H-BA, and changes in the shape o f the Soret difference spectrum during rebinding can be explained by ' 'kinetic hole burning''. That is, at sufficiently high viscosity the m ultiexponential ''low temperature'' rebinding of MbCO can be observed at all T, as predicted by Ansari et al. (Science 1992, 256, 1796). Mor eover, the average geminate rate predicted from the observed rate dist ribution is similar to 2500 times larger than the geminate rate in the completely relaxed protein in aqueous solution (Ansari et al Biochemi stry 1994, 33, 5128). Thus, we have shown that high solvent viscosity prevents both interconversion of conformational substates and function ally important relaxation in the interior of the protein, independent of T.