Compressibility of the heme pocket of substrate analogue complexes of cytochrome P-450cam-CO. The effect of hydrostatic pressure on the Soret band.

Abstract

The effect of hydrostatic pressure on the electronic absorption spectrum of the carbon monoxide complex of cytochrome P-450cam (CYP101) in the presence of various substrates was studied. With increasing pressure the wavenumber of the Soret band in the cytochrome P-450-CO complex shifts linearily to lower values (red-shift) and the half-width increases (broadening). The microscopic theory of solvent-solute interaction discussed by Laird and Skinner is used to explain the observed pressure effects. According to this theory, the slope of the red-shift of the Soret band is related to the compressibility of the chromophore environment, that is the heme moiety of the hemoproteins. It was found that the slope of the red-shift and the slope of the broadening of the Soret band for the CO complex in the presence of various substrate analogues increase with the decrease of the initial high-spin content at 0.1 MPa in the oxidized state. Variation of the high-spin content reflects the changes in the number of water molecules and/or changes in the polarity of the heme environment. The higher compressibility of the cytochrome P-450 complexes with the substrate analogues, which induce a lower degree of the high-spin content in the oxidized protein, is explained by the ability of the water molecules in the heme moiety to transmit the pressure effect on the protein structure to the heme chromophore. Therefore, a larger pressure-induced red-shift of the Soret band in the CO complex of cytochrome P-450cam might indicate a higher water content in the heme environment.