Stabilization of P4502B4 by its association with P450 1A2 revealed by high-pressure spectroscopy

We studied the effect of intermolecular interactions between cytochromes P4 50 1A2 (CYP1A2) and 2B4 (CYP2B4) on the barotropic inactivation of the ferr ous carbonyl complexes of the hemoproteins. When taken separately, these he moproteins reveal quite distinct barotropic behavior. While the 2B4(Fe2+)-C O complex is very sensitive to hydrostatic pressures and undergoes P450 --> P420 transition at rather low pressures (P-1/2 = 297 MPa, Delta V-0 = -61 ml/mol), the 1A2(Fe2+)-CO is extremely resistant to barotropic inactivation . Only about 8% of the 1A2 was exposed to pressure-induced P450 --> P420 tr ansition (P-1/2 = 420 MPa, Delta V-0 = -28 ml/mol). The formation of the mi xed oligomers of 2B4 and 1A2 was found to have a dramatic effect on the bar otropic behavior of 2B4. In the heterooligomers of 1A2 and 2B4, the 2B4 hem oprotein appears to be largely protected from barotropic inactivation. In 1 :1 mixed oligomers no more than 25% of the total P450 content undergoes P45 0 --> P420 inactivation with the molar reaction volume value (Delta V-0 = - 26 ml/mol) similar to those found for pure 1A2. Moreover, interactions betw een 1A2 and 2B4 results in a displacement of the Soret band of the ferrous carbonyl complex of CYP2B4 to shorter wavelength (from 451.3 to 448.4 nm) a nd largely strengthens the dependence of the Soret band wavenumber on hydro static pressure below 200 MPa. This effect suggests an important hydration of the CYP2B4 heme moiety in response to the interactions with CYP1A2. We d iscuss these results in terms of the hypothesis that the heterooligomerizat ion of cytochromes P450 in microsomes plays an important role in the contro l of the activity and coupling of the microsomal monooxygenase. (C) 2000 Ac ademic Press.