HIGH-PRESSURE-INDUCED TRANSITIONS IN MICROSOMAL CYTOCHROME-P450 2B4 IN SOLUTION - EVIDENCE FOR CONFORMATIONAL INHOMOGENEITY IN THE OLIGOMERS

Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a fu nction of benzphetamine concentration (0.05 divided by 2 mM) and state of aggregation of the hemoprotein in solution, Application of factor analysis to the spectral changes in the Soret region allowed us to res olve two particular pressure-induced processes in 2B4 oligomers, The f irst process was identified as the conversion of the low-spin P450 int o the P420 state, At 25 degrees C it was followed by decay (bleaching) of about 50% of the newly formed P420, The second process was a press ure-induced high- to low-spin shift, Both transitions were reversible, except the hemoprotein bleaching, The amplitude of the P450 --> P420 transition accounted for 67 +/- 5% of the total hemoprotein content, F urthermore, the fraction of the hemoprotein exposed to spin equilibriu m was not affected by the P450 --> P420 conversion and was estimated t o be only about 31 +/- 5% of the total hemoprotein content, After the dissociation of the oligomers by 0.2% Triton N-101, the inhomogeneity vanished: 95% of the monomers were involved in the P450 --> P420 trans ition (Delta V degrees = -86 ml/mol) mel) followed by intense bleachin g of the hemoprotein, This agrees with our earlier observations on the reduced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers, The parameters of the P450 --> P420 conversion (Delta V degrees = -32 ml/mol, P-1/2 = 15 60 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus benzphetamine concentration sh ows this transition to be caused mainly by changes in the spin equilib rium of substrate-bound (Delta V degrees = -49 ml/mol) and substrate-f ree (Delta V degrees = -21 ml/mol) hemoprotein, whereas the substrate binding step itself has a very weak. pressure dependency (Delta V degr ees = -8 ml/mol).