Geminate Rebinding and Conformational Dynamics of Myoglobin Embedded in a Glass at Room Temperature

• Published in: Journal of physical chemistry (1952) 1996-07, Vol.100 (29), p.12008-12021
• Main Authors: Hagen, Stephen J, Hofrichter, James, Eaton, William A
• Format: Article
• Language: English
• ISSN: 0022-3654; 1541-5740
• DOI: 10.1021/jp960219t

Below the glycerol/water glass transition (∼180 K), myoglobin exhibits distributed geminate rebinding kinetics as a result of “frozen” conformational substates (Austin et al. Biochemistry 1975, 14, 5355). As the temperature is increased through the solvent glass transition, 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 barriers to conformational change (Steinbach et al. Biochemistry 1991, 30, 3988). Using time-resolved spectroscopy with nanosecond lasers, we have studied ligand rebinding in sperm whale MbCO embedded in a glass at room temperature. Over a wide temperature range T = 105−297 K, the kinetics of rebinding are well characterized by the same inhomogeneous distribution g(H BA) of enthalpy barriers H BA, and changes in the shape of the Soret difference spectrum during rebinding can be explained by “kinetic hole burning”. That is, at sufficiently high viscosity the multiexponential “low temperature” rebinding of MbCO can be observed at all T, as predicted by Ansari et al. (Science 1992, 256, 1796). Moreover, the average geminate rate predicted from the observed rate distribution is ∼2500 times larger than the geminate rate in the completely relaxed protein in aqueous solution (Ansari et al. Biochemistry 1994, 33, 5128). Thus, we have shown that high solvent viscosity prevents both interconversion of conformational substates and functionally important relaxation in the interior of the protein, independent of T.