A CENTRAL ROLE FOR WATER IN THE CONTROL OF THE SPIN-STATE OF CYTOCHROME P-450(SCC)

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-450(scc) (SCC), the enzyme which initiates steroid biosynthesis by cleaving the side chain of cholesterol. The co nformation found at the lowest temperatures (form A) displays a six-li gand high-spin heme iron [Hildebrandt, P., Heibel, G., Anzenbacher, P. , Lange, R., Kruger, V. & Stier, A. (1994) Biochemistry 33, 12920-1292 9], 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 diff er in the mean environment of the tryptophan residues, which was more polar in the A form. The structural role of water in these two conform ations was investigated using the pressure-jump technique under variou s pH, temperature and osmotic-stress conditions. Applying hydrostatic pressure to SCC induced very slow (tau >30 min) biexponential relaxati on kinetics corresponding to the high-spin to low-spin transition, Ana lysis 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 molecul es) between the B and the A conformers and protonation of their respec tive high-spin states. The sixth ligand of the high-spin form in the A conformer involves a water molecule and an unknown constraining struc ture.