TY - JOUR T1 - ON THE MECHANISM OF HYDROXYLATION REACTIONS CATALYZED BY CYTOCHROME P-450 JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 92 LP - 97 VL - 1 IS - 1 AU - MINOR J. COON AU - HENRY W. STROBEL AU - RODNEY F. BOYER Y1 - 1973/01/01 UR - http://dmd.aspetjournals.org/content/1/1/92.abstract N2 - The mechanism of drug demethylation was studied in the reconstituted liver microsomal enzyme system with the aid of highly purified enzymes which catalyze the formation and decomposition of superoxide. The results obtained show that superoxide is generated by NADPH-cytochrome P-450 reductase in the presence of NADPH and oxygen, and that agents such as superoxide dismutase and Tiron, which are known to decompose superoxide, inhibit cytochrome P-450-catalyzed drug hydroxylation. Under similar conditions neither catalase nor heat-inactivated dismutase causes an inhibition. A scheme is presented to show the possible role of superoxide in microsomal hydroxylation reactions with the understanding that it represents a working hypothesis. Several cautionary points should be emphasized at this state of our investigations on this free radical species. 1. The results obtained do not distinguish whether superoxide serves as an essential electron donor or as an activated form of oxygen. Thus, its role could be at one or more of three steps: transfer ofthe first electron, resulting in reduction of cytochrome P-450 to the ferrous state; attack on the substrate with oxygen insertion; or transfer of the second electron, which accompanies substrate hydroxylation with reduction of an oxygen atom to water. 2. Superoxide is a labile free radical species and, therefore, may generate or be in equilibrium with some other species, such as singlet molecular oxygen, which serves as an activated form of oxygen. 3. The results presented were obtained with column P-450, which couples to a varying extent with artificial or substitute reducing systems (xanthine + xanthine oxidase; photoilluminated riboflavin + methionine; or bromelinor steapsin-solubilized NADPH-cytochrome c reductase) as well as with the detergent-solubilized P-450 reductase in catalyzing drug hydroxylation. This coupling is completely inhibited by superoxide dismutase. In sharp contrast, A.S. P-450 couples only with the detergent-solubilized P-450 reductase in catalyzing drug hydroxylation, and the reaction is not readily inhibited by the dismutase. The scheme presented is in accord with all of the results thus far obtained with the reconstituted liver microsomal enzyme system, including the known stoichiometry of the hydroxylation reaction. Copyright © 1973 by The American Society for Pharmacology and Experimental Therapeutics ER -