Structural changes in cytochrome P-450cam effected by the binding of the enantiomers (1R)-camphor and (1S)-camphor.
A comparative study of the enantiomeric substrate [(1R)-camphor- and (1S)-camphor)-bound cytochrome P-450cam concerns the spin-state equilibrium, substrate dissociation, the thermal unfolding of the protein structure, and the subconformer equilibria observed in the infrared spectra of the carbon monoxide (CO) complex of cytochrome P-450cam. 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 measured using the high pressure technique is smaller by 16 +/- 9 cm3/mol for (1S)-camphor-bound P-450cam compared to the (1R)-camphor-bound P-450cam, which might indicate a higher water content in the protein and in the heme environment in the (1S)-camphor complex. The half-transition temperature of the thermal unfolding of 53.8 degrees C for the (1S)-camphor-bound oxidized cytochrome P-450cam is one degree lower than the value for the (1R)-camphor-bound protein (54.8 degrees C). In the reduced, CO-bound form of cytochrome P-450cam at 290 K the (1S)-camphor complex reveals another CO stretch vibration population distribution with slightly higher frequencies [1940.2 cm-1 (major band) and 1946.3 cm-1 (minor band)] compared to the (1R)-camphor complex [1939.7 cm-1 (major band) and 1930 cm-1 (minor band)]. A loosening 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)-camphor, therefore the increased water accessibility, and the weaker contact 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.
Max-Delbrück-Centrum für Molekulare Medizin Berlin-Buch, Berlin, Germany.
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Schulze
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Hoa
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Helms
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Wade
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Jung
D001665:Binding Sites; D002164:Camphor / Q000737:chemistry*; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D002412:Cations; D004578:Electron Spin Resonance Spectroscopy; D005990:Glycerol / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006874:Hydrostatic Pressure; D007700:Kinetics; D009038:Motion; D011487:Protein Conformation; D011489:Protein Denaturation; D016958:Pseudomonas putida / Q000201:enzymology; D011994:Recombinant Proteins; D012997:Solvents / Q000737:chemistry; D013055:Spectrophotometry, Infrared; D013237:Stereoisomerism; D013696:Temperature; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D002412:Cations; D011994:Recombinant Proteins; D012997:Solvents; D014867:Water; D006418:Heme; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D005990:Glycerol