A Central Role for Water in the Control of the Spin State of CytochromeP‐450scc

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Abstract
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 (τ >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.