Conformational Change and Activation of Cytochrome P450 2B1 Induced by Salt and Phospholipid 1 Chul-Ho Yun,* ,2 Taeho Ahn,† and F. Peter Guengerich‡ *Department of Biochemistry, Pai-Chai University, Taejon 302-735, Korea; †Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon 305-701, Korea; and ‡Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received March 23, 1998, and in revised form May 8, 1998 A stimulatory effect of increased salt concentration on the enzymatic activity of rat liver microsomes and a reconstituted system containing cytochrome P450 (P450) 2B1 and NADPH-P450 reductase was seen. Structural change of P450 2B1 accompanying the salt- induced increase in its enzyme activity was investi- gated by circular dichroism, fluorescence spectros- copy, and absorption spectroscopy. It was found that the salt increased -helix content of P450 2B1 in the presence as well as in the absence of a phospholipid. Intrinsic fluorescence emissions also increased with increasing salt concentration. The low-spin iron con- figuration of P450 2B1 shifted toward the high-spin configuration in response to the increased salt concen- tration. It was found that the activity increase of P450 coincides with the raised -helix content. The pres- ence of phospholipid magnified this effect. It is pro- posed that the interaction with salts and phospholipid molecules surrounding P450 2B1 in the endoplasmic reticulum is important for a functional conformation of P450 2B1 in a monooxygenase system including NADPH-P450 reductase. © 1998 Academic Press Key Words: cytochrome P450 2B1; salt; phospholipid; conformation; activity. The microsomal monooxygenase system oxidized a variety of endogenous and xenobiotic compounds (1). This enzyme system includes cytochrome P450 3 (also termed heme-thiolate protein P450 by the Enzyme Commission, EC 1.14.14.1) (2), NADPH-P450 reduc- tase, and phospholipids. Cytochrome b 5 and NADH- cytochrome b 5 reductase may also contribute to the electron flow (3). P450-dependent activities can be re- constituted by mixing P450, NADPH-P450 reductase, and PC (4, 5). P450 and NADPH-P450 reductase seem to be distributed randomly on the plane of membranes, and they interact through lateral diffusion (6, 7). How- ever, the organization of constituent proteins in phos- pholipid membranes and their mechanism of interac- tion are not fully understood yet. P450 and NADPH- P450 reductase have been reported to interact by forming a functional complex for electron transfer (8). On the other hand, P450 is present in the membrane in large excess over the reductase, with the molar ratios ranging from 10:1 to 25:1 depending on treatment with inducers (9). Since NADPH-P450 reductase is the lim- iting component in microsomes, different P450 en- zymes must compete for the available reductases. It 1 This research was supported in part by G7 Research Project Grant from the Ministry of Science and Technology, Republic of Korea (C.-H. Y.) and United States Public Health Service (USPHS) Grants R35 CA44353 and P30 ES00267 (F.P.G.). 2 To whom correspondence should be addressed: Department of Biochemistry, Pai-Chai University, 439-6, Doma-dong, Seo-ku, Tae- jon 302-735, Korea. Tel: (82-42)-520-5612; Fax: (82-42)-533-7354; E-mail: chyun@woonam.paichai.ac.kr. 3 Abbreviations used: P450, cytochrome P450; CD, circular di- chroism; PC, phosphatidylcholine; PE, phosphatidylethano- lamine; DDPC, L--1,2-didecanoylphosphatidylcholine (C10:0); DUPC, L--1,2-diundecanoylphosphatidylcholine (C11:0); DLPC, L--1,2-dilauroylphosphatidylcholine (C12:0), DMPC, L--1,2- dimyristoylphosphatidylcholine (C14:0); DPPC; L--1,2-dipalmi- toylphosphatidylcholine (C16:0); DSPC L--1,2-distearoylphos- phatidylcholine (C18:0); DAPC, L--1,2-diarachidoylphosphatidyl- choline (C20:0); DLPE, L--1,2-dilauroylphosphatidylethanolamine (C12:0); DMPE, L--1,2-dimyristoylphosphatidylethanolamine (C14: 0); DPPE, L--1,2-dipalmitoylphosphatidylethanolamine (C16:0); DSPE, L--1,2-distearoylphosphatidylethanolamine (C18:0): DMPG, L--1,2-dimyristoylphosphatidylglycerol (C14:0); DPPG, L--1,2- dipalmitoylphosphatidylglycerol (C16:0); DSPG, L--1,2-distearoyl- phosphatidylglycerol (C18:0); PS, bovine brain phosphatidylserine; PA, egg yolk phosphatidic acid; PI, bovine liver phosphatidylinositol; PG, phosphatidylglycerol; CL, bovine heart cardiolipin; PROD, 7-pentoxyresorufin O-deethylation. 0003-9861/98 $25.00 229 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 356, No. 2, August 15, pp. 229 –238, 1998 Article No. BB980759