PROBING HEME PROTEIN CONFORMATIONAL EQUILIBRATION RATES WITH KINETIC SELECTION

Double-pulse flash photolysis experiments on solutions of carbonmonoxy myoglobin (MbCO) are used to determine the time scale for protein conf ormational averaging. The interconversion times for transitions betwee n the ''open'' and ''closed'' subpopulations of MbCO are found to be 1 0(-6)-10(-4) s, depending on solvent composition and temperature. In a queous solution at 273 K, the interconversion rate is found to be 1.4 x 10(6) s. Since the interconversion rate is comparable to or slower t han the geminate rebinding rate, we describe the geminate phase of the kinetics as a superposition of contributions from the open and closed states. Although geminate kinetics remain intrinsically nonexponentia l for both open and closed states near room temperature, we find that substates within these two subpopulations interconvert more rapidly th an the geminate rebinding. These observations cannot be explained by a superposition of contributions from a quasicontinuous conformational distribution (Steinbach et al., 1991) and are probably due to the long -time tail of the relaxation of the protein (Tian et al., 1992). Bimol ecular rebinding takes place at a statistically averaged rate, since t he interconversion and relaxation rates are faster than the bimolecula r kinetics. The geminate and bimolecular kinetics are analyzed quantit atively as a function of pH using this approach and the spectroscopica lly determined populations of the open and dosed states. The analysis accounts for the observed kinetics and also successfully predicts the kinetic response observed in the double-pulse experiments. In aqueous solution at 273 K, the geminate amplitudes and rates are found to be I -g(0) = 32% and k(g)(0) = 1.3 x 10(7) s(-1) for the open state and I-g (1) = 9.3% and k(g)(1) = 1.4 x 10(6) s(-1) for the closed state. In 75 % glycerol solution at 264 K, the dominant component of the geminate r ebinding is characterized by I-g1(0) = 89% and k(g1)(0) = 3.1 x 10(6) s(-1) for the open state and I-g1(1) = 26% and k(g1)(1) = 3.1 x 10(6) s(-1) for the closed state. The fact that the interconversion rate is comparable to the geminate rate of the closed state in aqueous solutio n is consistent with the idea that the open state provides an importan t pathway for ligand escape from (or entry to) the heme pocket (Tian e t al., 1993). The increased viscosity of 75% glycerol solution delays the closed --> open interconversion until the end of the geminate phas e, which forces the ligand to find alternative pathways to the solutio n. This observation, in conjunction with the near equivalence of the g eminate rates for the open and closed states in 75% glycerol solution, suggests that the solvent composition fundamentally alters the protei n-ligand dynamics.