STRUCTURAL-CHANGES IN CYTOCHROME P-450(CAM) EFFECTED BY THE BINDING OF THE ENANTIOMERS (1R)-CAMPHOR AND (1S)-CAMPHOR

A comparative study of the enantiomeric substrate [(1R)-camphor- and ( IS)-camphor)-bound cytochrome P-450(cam) concerns the spin-state equil ibrium, substrate dissociation, the thermal unfolding of the protein s tructure, and the subconformer equilibria observed in the infrared spe ctra of the carbon monoxide (GO) complex of cytochrome P-450(cam). The behavior of the different conformational equilibria in dependence on temperature, pressure, pH-value, cosolvent, and cation binding led us to suggest that (1S)-camphor is more loosely and less optimally bound in the heme pocket, which facilitates the access of solvent molecules into the heme-iron environment, The spin reaction volume difference me asured using the high pressure technique is smaller by 16 +/- 9 cm(3)/ mol for (1S)-camphor-bound P-450(cam) compared to the (1R)-camphor-bou nd P-450(cam), which might indicate a higher water content in the prot ein and in the heme environment in the (IS)-camphor complex, The half- transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450(cam) is one degree lo wer than the value for the (IR)-camphor-bound protein (54.8 degrees C) , In the reduced, GO-bound form of cytochrome P-450(cam) at 290 K the (1S)-camphor complex reveals another CO stretch vibration population d istribution with slightly higher frequencies [1940.2 cm(-1) (major ban d) and 1946.3 cm(-1) (minor band)] compared to the (1R)-camphor comple x [1939.7 cm(-1) (major band) and 1930 cm(-1) (minor band)], A looseni ng of the contact between the iron-bound CO ligand and amino acids of the I-helix, probably induced by compensating effects of the increased water content, is suggested. Assuming the carbon monoxide complex as a model for the dioxygen complex, the more loosened binding of (1S)-ca mphor, therefore the increased water accessibility, and the weaker con tact of the iron ligand to the I-helix might explain the higher amount of uncoupling of the cytochrome P-450 reaction cycle compared to that when (1R)-camphor is used as substrate.