ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 275, No. 2, December, pp. 429-439,1989 Secondary Structure and Membrane Topology of Cytochrome P45Os VADIM E. TRETIAKOV,*,’ KIRILL N. DEGTYARENKO,? VALENTIN Yu. UVAROV,? AND ALEXANDER I. ARCHAKOVt *Institute of Physical-Chemical Medicine, Malaya Pirogovskaya la, Moscow 119828, USSR, and t2nd Moscow Medical Institute, Ostrovityanova 1, Moscow 117437, USSR Received February 24,1989, and in revised form June 20,1989 The secondary structure prediction of 19 microsomal cytochrome P45Os from two different families was made on the basis of their amino acid sequences. It was shown that there is structural similarity between the heme-binding sites in these enzymes and those in the bacterial P450cam. An average predicted secondary structure of cytochrome P450 proteins with 70% accuracy contains about 46% a-helices, 12% ,&sheets, 9% p- turns, and 33% random coils. In the region of residues 35-120 in microsomal P45Os two adjacent pap-units (the Rossmann domain), were recognized and may be available to interact with the NADPH-cytochrome P450 reductase. Using the procedure for identifi- cation of hydrophobic and membrane-associated a-helical segments, only one N-termi- nal transmembrane anchor was predicted. Also the heme-binding site may include the surface-bound helix. A model for vertebrate microsomal P45Oshaving an amphipathic membrane protein located on the cytoplasmic side of the endoplasmic reticulum mem- brane, with their active center lying outside or on the bilayer border, is proposed. 8 19x9 Academic Press. Inc. Cytochrome P45Os represent a super- family of numerous hemoprotein monoox- ygenases that play an important role in the metabolism of a wide variety of endoge- nous and exogenous substrates. The most studied forms come from mammals and bacteria. In bacteria, the known P450 pro- teins are soluble, while in vertebrates they are membrane bound. The camphor 5-exo- monooxygenase from Pseudomonas putida has been crystallized, and the three-di- mensional structure is known to a resolu- tion of 1.6 A (1, 2). No three-dimensional structures are known for the membrane- bound forms; however, more than 100 dis- tinct vertebrate P450 amino acid se- quences have been determined. These se- quences contain information that can be used to predict the secondary structure and membrane topology of cytochrome P450. Predictions of cytochrome P450 second- ary structure by the method of Chou and 1 To whom correspondence should be addressed. Fasman (3) were made by Tarr et al. (4) for rabbit P450 form 2 and by Gotoh et al. (5) for rat P450b isozyme. It should be noted, though, that the method of Chou and Fas- man as well as some other predictive meth- ods is based on soluble globular protein secondary structure data. Therefore, ap- plication of this method to membrane pro- teins may not be entirely correct. Since no P-structures completely im- mersed in the lipid have been found in membrane proteins, the transmembrane segments are more likely to have a helical conformation. Thus, the accuracy of the prediction of the distribution of cY-helices has a significant effect on the prediction of protein membrane topology. Nelson and Strobe1 (6) analyzed 33 amino acid sequences of vertebrate cyto- chrome P45Os and the sequence of bacte- rial cytochrome P450cam. They studied the content of potential transmembrane re- gions corresponding to the hydrophobicity scales of Engelman et al. (7) and Kyte and 429 0003-9861/89 $3.00 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.