Dynamics of protein-bound water in the heme domain of P450BM3 studied by high-pressure spectroscopy: comparison with P450cam and P450 2B4.

Pressure-induced transitions in the heme domain of cytochrome P450BM3 (P450BMP) were studied versus the concentration of palmitic acid. An increase in hydrostatic pressure causes a high- to low-spin shift and subsequent P450 to P420 transition. Conversion of P450BMP to P420 is associated with important conformational and hydration changes of the protein. Treating the pressure-induced changes in the high-spin content in P450 in terms of the four-state model of spin transitions and substrate binding, we evaluated and compared the barotropic parameters of these transitions for P450MBP, P450cam, and P450 2B4 (2B4). In the current study, the pressure-induced transitions in P450cam were reinvestigated versus the concentration of camphor. The interactions of 2B4 and P450BMP with their substrates (benzphetamine and palmitic acid) were accompanied by larger changes in the partial volume of the proteins (+267 and +248 mL/mol, respectively) than the interactions of P450cam with camphor (+106 mL/mol). For 2B4 and P450BMP, substrate binding apparently requires hydration of regions outside the active site. The reaction volumes of the low- to high-spin transitions of the substrate-free cytochromes (20-23 mL/mol) are consistent with the displacement of one water molecule. The volume changes in the high- to low-spin transition of the substrate-bound P450cam, 2B4, and P450BMP (-90, -49, and -16 mL/mol correspondingly) reveal a linear relationship with DeltaG degrees of the spin transition, suggesting that modulation of the spin state by substrate binding is driven by a common mechanism in all three heme proteins.