A central role for water in the control of the spin state of cytochrome P-450scc.
A previous thermodynamic study [Lange, R., Larroque, C. & Anzenbacher, P. (1992) Eur. J. Biochem. 207, 69-73] demonstrated two conformations (A and B) of cytochrome P-450scc (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The conformation found at the lowest temperatures (form A) displays a six-ligand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P., Lange, R., Krüger, V. & Stier, A. (1994) Biochemistry 33, 12920-12929]. Analytical centrifugation shows that the oligomeric composition of SCC is the same for the A and the B conformers. However, as revealed by fourth-derivative ultraviolet spectroscopy, the two conformers differ in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conformations was investigated using the pressure-jump technique under various pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxation kinetics corresponding to the high-spin to low-spin transition. Analysis of the activation volumes suggested a dissociative mechanism for the A conformer (+45 ml/mol), and an associative mechanism for the B conformer (-39 ml/mol). Applying osmotic stress to the A form changed its kinetic characteristics to those of the B form. These results are consistent with a model comprising a solvent intake (ten water molecules) between the B and the A conformers and protonation of their respective high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining structure.
Bancel
F
F
INSERM U 128, Montpellier, France. bancel@crbm.cnrs-mop.fr
Bec
N
N
Ebel
C
C
Lange
R
R
D002498:Centrifugation; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009997:Osmotic Pressure; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature; D014867:Water / Q000737:chemistry*
D014867:Water; D002786:Cholesterol Side-Chain Cleavage Enzyme