ACTIVE-SITE ANALYSIS OF P450 ENZYMES - COMPARATIVE MAGNETIC CIRCULAR-DICHROISM SPECTROSCOPY

Recent structural studies indicate that the substrate-and O-2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (C YP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C. A, Hasemann et al., J. Mel. Biol. 236, 1169, 199 4). In contrast, the distal pockets of P450terp and P450BMP (CYP102 he me domain; Bacillus megaterium) are more closely similar, including na vel hydrogen-bonding interactions between the distal H2O ligand and th e I helix (C. A. Hasemann et al., Structure, 3, 41-62, 1995). To evalu ate the significance of these differences, we have compared solution m agnetic circular dichroism (MCD) spectra of P450terp with spectra of o ther P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of m ammalian P450LM-2 than to those of P450cam. Upon substrate-binding, th e MCD spectra of ferric P450terp and all other thiolate-ligated heme s ystems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or o ther B-coordinate ferric heme systems. This intense negative MCD featu re thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough va ries between substrate-bound and substrate-free enzymes (despite the f act that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts o f the Soret MCD band between the substrate-bound and substrate-free fo rms of ferrous-CO-P450terp. No such observation has been made previous ly. Furthermore, the band positions for BOTH types of P450terp are red -shifted from known bands of ferrous-CO-P50cam. These data thus indica te a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on GO-binding rates and equilibrium constants. Comparative analysis of t he spectral properties of P450terp with MCD spectra of other P450 enzy mes, as well as with chloroperoxidase and NO synthase, demonstrates bo th the expected similarities and the significant differences that refl ect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous -CO-P450. Furthermore, testable structural predictions for P450-BM-1 a nd for the novel NO synthase enzyme (neither of which has been crystal lized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes. (C) 1997 Academic Press.