Chiral sulfoxidations catalyzed by rat liver cytochromes P-450.

Abstract

The chirality of sulfoxidation catalyzed by two cytochrome P-450 isozymes purified from phenobarbital-induced rat liver was studied by using 4-tolyl ethyl sulfide as a substrate. Both P-450 isoenzymes, termed PB-1 and PB-4, when reconstituted with purified rat liver NADPH-cytochrome P-450 reductase and cytochrome b5, generated 4-tolyl ethyl sulfoxide which was predominantly in the S-(-) configuration. In the case of isozyme PB-1, the sulfoxide was 79 +/- 1% S and was formed with a turnover of 41 min-1; with isoenzyme PB-4, sulfoxide, 84 +/- 1%, S, was formed at 31 min-1. In addition, PB-1-1 catalyzed oxygen transfer to the p-methyl group of the sulfide substrates to yield the (ethylthio)benzyl alcohol with a turnover of 6.8 min-1, corresponding to a sulfur:carbon oxygenation partition ratio of 6:1. Isozyme PB-4 was approximately 80-fold less efficient at catalyzing this carbon hydroxylation, giving a sulfur:carbon ratio of approximately 375:1. In the absence of cytochrome b5, turnover numbers were reduced to approximately 15% and 67% of the above values for PB-1 and PB-4, respectively, with no change in sulfoxide chirality. This fact, and the lack of improvement in chirality upon inclusion of scavengers for reactive oxygen species, suggests that the approximately 79-84% chirality observed for the sulfoxide product reflects an intrinsic lack of complete stereospecificity in these cytochrome P-450 catalyzed reactions. The enantiomeric composition of 4-tolyl ethyl sulfoxide generated in rat liver microsomal incubations was shown to reflect the relative contribution of cytochrome P-450 isozymes, which generate the S-(-) enantiomer preferentially, and of the flavin adenine dinucleotide (FAD) containing monooxygenase (EC 1.14.13.8), which as we have shown catalyzes (R)-(+)-sulfoxide formation [Light, D. R., Waxman, D. J. & Walsh, C. (1982) Biochemistry (preceding paper in this issue)]. Thus, the chirality of microsome-catalyzed sulfoxidation is shown to be modulated by factors which alter the relative participation of these by factors which alter the relative participation of these two liver monooxygenases, such as phenobarbital induction, inclusion of inhibitors or activators (metyrapone and n-octylamine), and variation in sulfide substrate concentration.