ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 320, No. 2, July 10, pp. 330-344, 1995 High-Pressure-Induced Transitions in Microsomal Cytochrome P450 2B4 in Solution: Evidence for Conformational Inhomogeneity in the Oligomers Dmitri R. Davydov,’ Eric Deprez, Gaston Hui Bon Hoa, Tatiana V. Knyushko,” Galina P. Kuznetsova,” Yakov M. Keen,* and Alexander I. Archakov” Znstitut de Biologie Physico-Chimique, ZNSERM U.310, Paris, France; and ‘“Znstitute of Biomedical Chemistry, Russian Academy of Medical Sciences, Moscow, Russia Received October 24, 1994, and in revised form April 27, 1995 Pressure-induced changes in ferric P450 2B4 (LM2) were studied as a function of benzphetamine concen- tration (0.05 + 2 mu) and state of aggregation of the hemoprotein in solution. Application of factor analysis to the spectral changes in the Soret region allowed us to resolve two particular pressure-induced processes in 2B4 oligomers. The first process was identified as the conversion of the low-spin P450 into the P420 state. At 25°C it was followed by decay (bleaching) of about 50% of the newly formed P420. The second process was a pressure-induced high- to low-spin shift. Both transi- tions were reversible, except the hemoprotein bleach- ing. The amplitude of the P450 --t P420 transition ac- counted for 67 + 5% of the total hemoprotein content. Furthermore, the fraction of the hemoprotein exposed to spin equilibrium was not affected by the P450 -, P420 conversion and was estimated to 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 transition (AV” = -86 ml/ mol) followed by intense bleaching of the hemoprotein. This agrees with our earlier observations on the re- duced carbonyl complex of P450 2B4 and suggests some conformational difference between subunits in P450 LM2 oligomers. The parameters of the P450 + P420 conversion (AV” = -32 ml/mol, Plls = 1560 bar) show no dependency on the substrate concentration. Analysis of the pressure-induced spin shift versus ben- zphetamine concentration shows this transition to be caused mainly by changes in the spin equilibrium of i Present address: Institute of Biological and Medical Chemistry, Russian Academy of Medical Science, 10 Pogodinskaya ul., 119832, Moscow, Russia. ‘To whom correspondence should be addressed. Fax: (33-l) 43-29- 80-88. E-mail: HUIBONHOQIBPC.FR. 330 both substrate-bound (AV” = -49 ml/mol) and sub- strate-free (AV” = -21 mYmo1) hemoprotein, whereas the substrate binding step itself has a very weak pres- sure dependency (AV” = -8 ml/mol). v 1995 Academic Press. Inc. In the past few years pressure-induced transitions in protein structure have been used successfully to study changes in the heme moiety of cytochromes P450,,,, and P45Oiin on substrate binding (l-6). Recently, we reported the first results of pressure-induced transi- tions in rabbit liver microsomal cytochrome P450 2B4 (7). This hemoprotein (also known as cytochrome P450 LM2) is denoted “2B4” in this paper.” Pressure-induced inactivation (P450 + P420 transition and disappear- ance of the hemoprotein-specific bands at high pres- sures) of the complex of the reduced hemoprotein with carbon monoxide was studied (7). This membrane hem- oprotein pressure-related behavior was certainly differ- ent from that of the soluble bacterial cytochromes P45OCXn and P450ii,. This difference is caused partly by the oligomeric structure of the microsomal hemo- protein in solution. The molecular masses reported by several authors for microsomal cytochromes P450 sug- gest the size of the aggregates to be in the range of 6-10 protomer molecules (8-12). Electron microscopy studies of P450 2B4 (13) and P450 lA2 (14) oligomers show them to be hexamers. Within the membranes, microsomal P45Os were also proven to form oligomers, which are believed to be hexamers as well (15-18). a Abbreviations used: 2B4, cytochrome P450 2B4 (LM2) from rabbit liver microsomes; P450 and P420, cytochrome P450 2B4 in the P450 and P420 states, respectively; P450,,, and P450,,, high-spin and low- spin states of the cytochrome; PCA, principal component analysis. 0003-9861195 $12.00 Copyright ~3 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.