Treatment of purified cytochrome P-450 LM2 and its liposome-bound form with hydrogen peroxide led to complete destruction of the P-450 heme. The apoenzyme thus produced could be reconstituted to catalytically active cytochrome P-450 by incubation with hemin, the reconstitution efficiency being 50% for the soluble enzyme and 80% for the liposome-bound enzyme. The removal of heme from the soluble hemoprotein resulted in a 3-fold decrease in the efficiency of its incorporation into sonicated liposomes. The contents of 5 secondary structure forms in the native, apoand reconstituted holoenzymes were estimated from their circular dichroism spectra. It was thus found that the helix content increased from 34% to 60% upon removal of the heme from the native enzyme. We suggest that the increase in the helix content leads to a reduction of the incorporation efficiency into liposomal membranes.

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Membrane-bound sites of cytochrome P-450 2B4 (LM2) were determined by means of two different methods, photoactivated binding of membrane phospholipids to the protein and epitope mapping by antibodies. Phospholipids bearing photoreactive labels at different distances from the their polar 'head' were used in the former case. Phosphatidylcholine labelled at the apolar end of the fatty acid chain bound only to the N-terminal region of the hemoprotein. Other phospholipids labelled nearer to the head group bound not only to the N-terminus but also to the segments 273-314 and 427-491. Epitope mapping of the domain next to the N-terminus (residues 21-119) of the isolated hemoprotein was performed with the help of a peptide-scanning method, a programmable peptide synthesis on pins followed by ELISA testing with the polyclonal antiserum against cytochrome P-450 2B4. This domain was shown to possess a considerable density of sites with high antigenic activity. No membrane-penetrating part of this domain was found except for the fragment 1-21. A model of structure of P-450 2B4 was computed by comparison with the structure of cytochrome P-450,, on the basis of an alignment of 47 cytochromes P-450 with the former hemoprotein. Major parts of the protein sequences photoreacting with the phospholipid probes, but not the antibody-reactive epitopes of the region 21 -119, are located at the membrane-facing side in this model Though cytochromes P-450 are the most thoroughly studied membrane enzymes, there is no agreement on their topology in the lipid bilayer. At present there are several models of the membrane orientation of microsomal cytochrome P-450 [l-61 constructed on the basis of various methods of polypeptide chain-structure prediction or some experimental data [7-111. The aim of our investigation was to examine these models by analysis of the interaction of photoreactive phospholipids bearing the photolabel at different distances from their polar 'heads' with cytochrome P-450 2B4 in proteoliposomes and by surface epitope mapping of the 119 N-terminal amino acid residues in the solubilized protein with the help of the peptide-scanning technique (PEPSCAN) [ 121. MATERIALS AND METHODS MaterialsCytochrome P-450 2B4 was obtained from phenobarbital-induced rat liver microsomes [ 131. Proteoliposomes were prepared as described earlier [14]. Photoreactive lipid was added to the solution of egg phosphatidylcholine, a phosphoCorrespondence to V. Y. Uvarov, Institute Biomedical Chemis-

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