HIGH-PRESSURE FLASH-PHOTOLYSIS STUDY OF HEMOPROTEIN - EFFECTS OF SUBSTRATE-ANALOGS ON THE RECOMBINATION OF CARBON-MONOXIDE TO CYTOCHROME P450(CAM)

The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450(CAM) (P450(CAM)) at 293 K have been studied as a function of hydrostatic pressure (0.1-200 MPa) by means of flash photolysis. At 0.1 MPa, the association rate constant (k(on)) for substrate-free P450(CAM) is 8.5 x 10(6) M(-1) s(-1). Measurements as a function of pressure lead to a determination of the activation volume (Delta V-double dagger) of +4 cm(3) mol(-1) for substrate-free protein. This positive Delta V-double dagger is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative Delta V-double dagger values [Adachi, S., & Morishima, I. (1989) J. Biol. Chem. 264, 18896-18901; Unno, M., Ishimori, K., & Morishima, I. (1990) Biochemistry 29, 10199-10205]. The binding of d-camphor and some camphor analogues (d-fenchone, 3-endo-bromocamphor, and 3,3,5,5-tetramethylcyclohexanone) into the heme pocket strongly influences the kinetics, i.e., k(on) is reduced ((1-10) x 10(5) M(-1) s(-1)) and Delta V-double dagger is altered to a negative value (-14 to -32 cm(3) mol(-1)). The negative Delta V-double dagger suggests that the effects of camphor and these camphor analogues are due to an increase in the iron-ligand bond formation barrier. On the other hand, the binding of adamantane and norcamphor does not affect the kinetics. This result is particularly surprising because both substrate analogues are located in the immediate vicinity of the CO binding site. Since both adamantane and norcamphor show high mobility in the heme active site, we conclude that a substrate fluctuation at the heme active site is an important determinant of the rate of the bond formation process.