Step-scan time-resolved FTIR spectroscopy of cytochrome P-450cam carbon monoxide complex: a salt link involved in the ligand-rebinding process.
Step-scan time-resolved Fourier transform infrared spectroscopy with a time resolution of 5 micros was applied to the carbon monoxide complex of cytochrome P-450cam (CYP101) to study the bimolecular ligand-rebinding process after flash photolysis. Spectral changes in the CO ligand stretch vibration band and in the protein amide I' band were monitored simultaneously. In substrate complexes having the camphor C-8, C-9, and C-10 methyl groups, rebinding of the ligand and the relaxation of the protein proceed at the same rate within experimental errors. For substrate complexes missing the methyl groups, the relaxation fo the protein tends to relax slightly faster than the CO ligand rebinding to the heme iron. compared to the (1R)-camphor and the camphane complex, the bimolecular rebinding rate constant for P-450 bound with substrates lacking the methyl groups are increased by a factor of 10-40. An unusual signal at about 1719 cm-1 was found in the difference spectrum of the photolyzed minus nonphotolyzed CO complex which has not ben reported for other heme proteins so far. This signal is strongly pronounced in wild-type P-450cam bound with (1R)-camphor or camphane and in the D251N mutant bound with (1R)-camphor. In contrast, substrate-free P-450 and the norbornane and norcamphor complexes reveal only a very weak signal or a changed band shape. On the basis of the crystal structure data, we suggest that this signal originates from the rearrangement of the hydrogen-bonding pattern or the protonation state of the salt link between Asp297, Arg299, and the heme propionate group.
Contzen
J
J
Max-Delbrück Center for Molecular Medicine Berlin-Buch, Berlin, Germany.
Jung
C
C
D001892:Camphanes / Q000378:metabolism; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D002248:Carbon Monoxide / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D009636:Norbornanes / Q000378:metabolism; D010782:Photolysis; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013379:Substrate Specificity
D001892:Camphanes; D008024:Ligands; D009636:Norbornanes; C067796:camphane; C055059:norcamphor; D002164:Camphor; D002248:Carbon Monoxide; D019475:Camphor 5-Monooxygenase