Unusual pressure effects on ligand rebinding to the human myoglobin Leucine 29 mutants.
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.
Uchida
T
T
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Kyoto 606-8501, Japan.
Ishimori
K
K
Morishima
I
I
D002248:Carbon Monoxide / Q000378:metabolism*; D006801:Humans; D006874:Hydrostatic Pressure; D007930:Leucine; D008024:Ligands; D008956:Models, Chemical; D009154:Mutation; D009211:Myoglobin / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D010100:Oxygen / Q000378:metabolism*; D010782:Photolysis
D008024:Ligands; D009211:Myoglobin; D002248:Carbon Monoxide; D007930:Leucine; D010100:Oxygen