Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods Eva Anzenbacherova a , Jiri Hudecek b , Daniel Murgida c , Peter Hildebrandt c , Ste ´phane Marchal d , Reinhard Lange d , Pavel Anzenbacher e, * a Institute of Medical Chemistry and Biochemistry, Faculty of Medicine, Palacky University, CZ-775 15 Olomouc, Czech Republic b Department of Biochemistry, Faculty of Sciences, Charles University, CZ-128 40, Prague 2, Czech Republic c Max-Volmer-Laboratory for Biophysical Chemistry, Technical University Berlin, 10623 Berlin, Germany d INSERM U710, University of Montpellier 2, 34095 Montpellier Cedex 5, France e Institute of Pharmacology, Faculty of Medicine, Palacky University, CZ-775 15 Olomouc, Czech Republic Received 26 July 2005 Available online 22 August 2005 Abstract Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Dav- ydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121–130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; CYP2E1; Active site; Resonance Raman; Absorption spectroscopy; High pressure Cytochrome P450 2E1 (CYP2E1) is an enzyme known to be expressed in liver as well as in extrahepatic tissues as, e.g., brain, lungs or lymphocytes. Its endoge- nous function remains to be elucidated, although there are clues indicating its involvement in lipid and glucose metabolism [1]. CYP2E1 belongs to one of the first liver microsomal cytochromes P450 isolated and its role in metabolism of relatively small xenobiotics (as ethanol, anesthetics, and solvents) as well as in activation of car- cinogens (nitrosamines) has been widely studied. In the absence of crystallographic data for this enzyme, discus- sions of structure–function relationships rely on models based on known structures of other CYP forms [2,3]. Active sites of liver microsomal CYP enzymes should be able to accommodate a variety of substrates, however maintaining a certain degree of specificity. Recent crys- tallographic analyses have shown that the volume of the active site, i.e., the space available for a substrate is not the only factor determining the ability of a substrate to be present in the active site. Surprisingly, the CYP3A4 enzyme crystal structure has been shown to possess a small active site, although the substrates of this enzyme are molecules with MW up to 1 kDa [4,5]. The key to understanding this seeming discrepancy may be in the flexibility of the enzyme active site. In fact, the apparent compressibility (determined from the redshift of the Sor- et absorption band with hydrostatic pressure [6]) of the CYP3A4 heme pocket in the presence of substrate has been shown to be relatively high among CYP enzymes 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.063 * Corresponding author. Fax: +420 58 563 2569. E-mail address: anzen@tunw.upol.cz (P. Anzenbacher). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 338 (2005) 477–482 BBRC