The effects of high pressure (1-2000 bar) on the spin state and substrate binding equilibria in cytochrome P-450 have been determined. The high-spin (S = 5/2) to low spin (S = 1/2) transition of the ferric hemoprotein was monitored by uv-visible spectroscopy at various substrate concentrations. Increasing hydrostatic pressure on a sample of substrate-bound cytochrome P-450 resulted in a decrease in the high-spin fraction as monitored by a Soret maxima at 391 nm and an increase in the low-spin 417-nm region of the spectrum. These pressure-induced optical changes were totally reversible for all pressures below 800 bar and were found to correspond to simple substrate dissociation from the enzyme. High levels of the normally metabolized substrate, d-camphor, corresponding to a 99.9% saturation of the hemoprotein active site (50 mM Tris-Cl, 100 mM KCl, pH 7.2) completely prevented the pressure-induced high-spin to low-spin transition that is observed at less than saturating substrate concentrations. A gradual increase in the formation of the inactive P-420 form of the cytochrome was noted if the pressure of the sample was increased above 800 bar. These pressure-linked spectral changes were used to determine the microscopic volume change accompanying substrate binding, which was found to be -47.0 +/- 2 ml/mol (pH 7.2) which represents a substantial change for a ligand dissociation reaction. The observed volume change for camphor binding decreases to -30.6 +/- 2 ml/mol at pH 6.0, suggesting the involvement of a linked proton equilibrium. Various substrate analogs of camphor induce varying degrees of low-spin to high-spin shift upon binding to ferric cytochrome P-450 (3). The volume changes for the dissociation of these substrates were very similar to those obtained with camphor. The conformational changes associated with a shift from high- to low-spin ferric iron appear to be small in comparison to the overall macroscopic changes in volume accompanying substrate binding to the enzyme.