Redox cycling of resorufin catalyzed by rat liver microsomal NADPH-cytochrome P450 reductase

Abstract

The O-dealkylation of 7-alkoxyresorufins to the highly fluorescent compound, resorufin (7-hydroxyphenoxazone), provides a rapid, sensitive, and convenient assay of certain forms of liver microsomal cytochrome P450. The results of this study indicate that NADPH-cytochrome P450 reductase catalyzes the reduction of resorufin (and the 7-alkoxyresorufins) to a colorless, nonfluorescent compound(s). The reduction of resorufin by NADPH-cytochrome P450 reductase was supported by NADPH but not NADH, and was not inhibited by dicumarol, which established that the reaction was not catalyzed by contaminating DT-diaphorase (NAD[P]H-quinone oxidoreductase). In addition to the rate of reduction, the extent of reduction of resorufin was dependent on the concentration of NADPH-cytochrome P450 reductase. The maintenance of steady-state levels of reduced resorufin required the continuous oxidation of NADPH, during which molecular O 2 was consumed. When NADPH was completely consumed, the spectroscopic and fluorescent properties of resorufin were fully restored. These results indicate that the reduction of resorufin by NADPH-cytochrome P450 reductase initiates a redox cycling reaction. Stoichiometric measurements revealed a 1:1:1 relationship between the amount of NADPH and O 2 consumed and the amount of H 2O 2 formed (measured fluorometrically). The amount of O 2 consumed during the redox cycling of resorufin decreased ~50% in the presence of catalase, whereas the rate of O 2 consumption decreased in the presence of Superoxide dismutase. These results suggest that, during the reoxidation of reduced resorufin, O 2 is converted to H 2O 2 via superoxide anion. Experiments with acetylated cytochrome c further implicated superoxide anion as an intermediate in the reduction of O 2 to H 2O 2. However, the ability of reduced resorufin to reduce acetylated cytochrome c directly (i.e., without first reducing O 2 to superoxide anion) precluded quantitative measurements of superoxide anion formation. Superoxide dismutase, but not catalase, increased the steady-state level of reduced resorufin and considerably delayed its reoxidation. This indicates that superoxide anion is not only capable of reoxidizing reduced resorufin, but is considerably more effective than molecular O 2 in this regard. Overall, these results suggest that NADPH-cytochrome P450 reductase catalyzes the one-electron reduction of resorufin (probably to the corresponding semiquinoneimine radical) which can either undergo a second, one-electron reduction (presumably to the corresponding dihydroquinoneimine) or a one-electron oxidation by reducing molecular O 2 to superoxide anion. The superoxide anion formed is then converted to hydrogen peroxide, either by a second, one-electron oxidation of reduced resorufin or by dismutation. In the accompanying paper, we show that the redox cycling of the 7-alkoxyresorufins by NADPH-cytochrome P450 reductase significantly affects their 0-dealkylation by purified isozymes of rat liver microsomal cytochrome P450.