Abstract
Using high pressure flash photolysis, we revealed that the side chain of Leu(29) controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu(29) mutants. As we previously reported (Adachi, S., Sunohara, N., Ishimori, K., and Morishima, I. (1992) J. Biol. Chem. 267, 12614-12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration. However, very slow CO bimolecular rebinding of the mutants implies that bond formation is still the rate-determining step. To gain further insights into effects of the side chain on ligand binding, we prepared some new Leu(29) mutants to measure the CO and O(2) rebinding reaction rates under high hydrostatic pressure. CO bimolecular rebinding in the mutants bearing Gly or Ser at position 29 was also decelerated upon pressurization, resulting in apparent positive activation volumes (DeltaV), as observed for O(2) binding. Based on the three-state model, we concluded that the increased space available to ligands in these mutants enhances the volume difference between the geminate and deoxy states (DeltaV(32)), which shifts the apparent activation volume to the positive side, and that the apparent positive activation volume is not due to contribution of the ligand migration process to the rate-determining step.
Highlights
In biophysical chemistry, hydrostatic pressure has long been used to affect dynamics, conformational equilibrium, and other properties of native states of proteins [1,2,3,4]
Using high pressure flash photolysis, we revealed that the side chain of Leu29 controls the reaction volume of the ligand migration process in myoglobin, which is the primary factor for the unusual activation volume of ligand binding in some Leu29 mutants
Chem. 267, 12614 –12621), CO bimolecular rebinding in the L29A mutant was unexpectedly decelerated by pressurization, suggesting that the rate-determining step is switched to ligand migration
Summary
Hydrostatic pressure has long been used to affect dynamics, conformational equilibrium, and other properties of native states of proteins [1,2,3,4]. Since the magnitude of activation volumes depends on conformational changes accompanied by reactions of proteins and reflects the specific interactions of the transition state with ligands, the volume profiles can provide us with a wealth of information on the dynamics of ligand binding processes. We found an anomalous pressure dependence of the CO rebinding rates for the myoglobin (Mb) L29A mutant [9] and cytochrome P450cam in the absence of camphor [12]. Both of them exhibited positive apparent activation volumes, which were interpreted in the sense that the rate-determining step was switched from the bond formation to the ligand diffusion process. The CO rebinding rate for the Mb L29A mutant was substantially slow (0.15 MϪ1 sϪ1) [9] compared with that for wild-type Mb (0.70 MϪ1 sϪ1), suggesting that the positive apparent activation volume
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