High-pressure flash photolysis study of hemoprotein: effects of substrate analogues on the recombination of carbon monoxide to cytochrome P450CAM.
The effects of camphor and camphor analogues on the CO recombination kinetics of ferrous cytochrome P450CAM (P450CAM) 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 (kon) for substrate-free P450CAM 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 not equal to) of +4 cm3 mol-1 for substrate-free protein. This positive delta V not equal to is interesting because the CO association reaction of various hemoproteins, such as myoglobin and hemoglobin, exhibit negative delta V not equal to 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., kon is reduced ((1-10) x 10(5) M-1 s-1) and delta V not equal to is altered to a negative value (-14 to -32 cm3 mol-1). The negative delta V not equal to 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Unno
M
M
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Ishimori
K
K
Ishimura
Y
Y
Morishima
I
I
D002164:Camphor / Q000031:analogs & derivatives*; D019475:Camphor 5-Monooxygenase; D002248:Carbon Monoxide / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005296:Ferrous Compounds / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism; D006874:Hydrostatic Pressure; D007700:Kinetics; D007834:Lasers; D008027:Light; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009211:Myoglobin / Q000378:metabolism; D010782:Photolysis; D016958:Pseudomonas putida / Q000201:enzymology*; D013816:Thermodynamics
D005296:Ferrous Compounds; D006454:Hemoglobins; D009211:Myoglobin; D002164:Camphor; D002248:Carbon Monoxide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019475:Camphor 5-Monooxygenase