One-electron reductive bioactivation of 2,3,5,6-tetramethylbenzoquinone by cytochrome P450

Abstract

Bioreductive activation of quinones in mammalian liver has generally been attributed to NADPH-cytochrome P450 reductase. However, in view of the 20–30-fold molar excess of cytochrome P450 over NADPH-cytochrome P450 reductase on the endoplasmic reticulum of the rat liver cell and the capability of cytochrome P450 to bind and reduce xenobiotics, it was considered of interest to investigate the possible role of cytochrome P450 in the bioreduction of quinones. In the present study, 2,3,5,6-tetramethyl-1,4-benzoquinone (TMQ) was chosen as a model quinone. First, TMQ was found to bind at the metabolic active site of phenobarbital (PB)-inducible cytochrome P450s of rat liver microsomes, indicating that TMQ is a potential substrate for cytochrome P450-mediated biotransformation. Second, with electron spin resonance, one-electron reduction of TMQ to a semiquinone free radical (TMSQ) was found to occur in these microsomal fractions. SK&F 525-A, a well-known inhibitor of cytochrome P450, strongly inhibited TMSQ formation in these subcellular fractions without affecting NADPH-cytochrome P450 reductase activity. One-electron reductive bioactivation of TMQ was further investigated with purified NADPH-cytochrome P450 reductase alone and in reconstituted systems of purified cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase. As measured by ESR, purified cytochrome P450-IIB1 in the presence of NADPH-cytochrome P450 reductase was able to reduce TMQ to TMSQ at a much greater rate than in the presence of NADPH-cytochrome P450 reductase alone. Reduction of TMQ was also investigated by measuring the initial rate of NADPH oxidation by TMQ under anaerobic conditions. Inhibitors of cytochrome P450, namely SK&F 525-A and antibodies against PB-inducible cytochrome P450s, caused a substantial decrease in reductive metabolism in PB-treated microsomes. These antibodies were also effective in the inhibition of TMQ-induced NADPH oxidation in a complete reconstituted system of equimolar concentrations of cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase, indicating that the reaction was specific for cytochrome P450-IIB1. Finally, initial rates of NADPH oxidation were determined in reconstituted systems containing varying amounts of NADPH-cytochrome P450 reductase and cytochrome P450-IIB1 to determine the contribution of either enzyme in the reduction of TMQ. As expected, NADPH-cytochrome P450 reductase was able to reduce TMQ to a small extent. However, reconstitution in the presence of increasing amounts of cytochrome P450-IIB1 (relative to NADPH-cytochrome P450 reductase) resulted in increasing rates of TMQ-induced NADPH oxidation. From these experiments and from reconstitution experiments with fixed amounts of cytochrome P450-IIB1 and varying amounts of NADPH-cytochrome P450 reductase, it was calculated that cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase contributed 1:1 to the reduction of TMQ. Interestingly, all experiments performed at an equimolar ratio of the two enzymes consistently resulted in a 4–5-fold enhancement of TMQ reduction by cytochrome P450-IIB1 as compared to NADPH-cytochrome P450 reductase alone. It is, therefore, suggested that cytochrome P450-IIB1 and NADPH-cytochrome P450 reductase act as one complex during one-electron reduction of TMQ to TMSQ under anaerobic conditions. Binding of TMQ to this preformed complex might enhance the efficiency of electron flux from NADPH-cytochrome P450 reductase to TMQ. In conclusion, this study demonstrates that in addition to NADPH-cytochrome P450 reductase, cytochrome P450-IIB1 contributes substantially to the reduction of TMQ in PB-induced rat liver microsomes under a relatively low oxygen tension. The role of cytochrome P450 in this type of reduction reaction may also be important for more complex biologically active quinoid compounds such as Adriamycin ® and mitomycin C.