The pressure dependence of the spin equilibrium in camphor-bound ferric cytochrome P-450.

The spin equilibrium of camphor-bound ferric cytochrome P-450 has been measured between 1-1000 bar (10(5)-10(8) Pa). Increasing pressure shifts the absorption spectrum from the high-spin form at 392 nm to the low-spin form at 417 nm. The molar volume change for the spin states delta V = -RT delta ln Ke/ delta P and the equilibrium coefficient Ke = [high spin]/[low spin] depend on the solvent conditions. At pH 5.6 the equilibrium coefficient at 1 bar, K1 = 0.5 and delta V = 312 cm3/mol. A sample with 10 mM KCl at pH 7 has K1 = 7.0 amd delta V = 52 cm3/mol. Solvent changes producing a larger K1 also result in a larger delta V which ranged over 16-74 cm3/mol. The correlation can be approximated as delta V = 36 + 18 log K1, which implies that there is a pressure, 3000 bar, for camphor-bound ferric cytochrome P-450 at 4 degrees C, at which the changes in delta V are compensated by the other thermodynamic parameters leaving Ke independent of the solvent conditions. Although the protein is not stable about 1000 bar for most sample conditions, the extrapolated log Ke versus pressure curves for all sample conditions intersect near 3000 bar. Camphor-bound cytochrome P-450 appears to be a rather flexible protein, having a low denaturing pressure, a large volume change, and a high sensitivity to the protein environment.