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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 highspin content in the oxidiz,ed 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.Keywords: cytochrome P-450; hydrostatic pressure; compressibilities; substrate binding; solute-solvent interaction.The effect of substrate binding on the high-spin/low-spin equilibrium of the heme iron in cytochrome P-450 is well documented for all cytochrome P-450 species (Schenkman and Greim, 1993) and is used as spectroscopic probe for determination of substrate binding constants. In addition, it was shown by Hui Bon Hoa and Marden (1982) that the spin transition of cytochrome P-450cam is extremely sensitive to physico-chemical conditions like pH, cations, temperature and pressure. A significant effect of substrate binding on the stretching mode of the CO ligand in cytochrome P-450cam-CO was recently shown in our laboratory (Jung et al., 1992a) and by Tsubaki and coworkers for cytochrome P-4SOscc (Tsubaki et al., 1992). Unno et al. (1994) influence of various substrate analogues on the recombination kinetics of the photodissociated CO ligand at different pressures. Furthermore, we have found that pressure has significant effect on the CO stretch frequency and population distribution between subconformers (Jung et a]., 199211; Schulze et al., 1994). It has been suggested that substrate mobility and water flux into the heme pocket are reflected in the changes of the CO ligand spectroscopic probe (Jung et al., 1992a,b;Schulze et al., 1994;Unno et al., 1994). These factors seem to be interrelated and control the degree of uncoupling of the cytochrome-P-450-catalyzed subs...

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Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties.