EVALUATION OF THE ROLE OF FREE HYDROXYL RADICALS IN THE CYTOCHROME-P-450-CATALYZED OXIDATION OF BENZENE AND CYCLOHEXANOL

  • 1 January 1985
    • journal article
    • research article
    • Vol. 13  (2) , 169-174
Abstract
The possible role of free hydroxyl radicals in the oxidation of cyclohexanol to cyclohexanone and of benzene to phenol was examined in a reconstituted system containing rabbit phenobarbital-incudible P-450LM2. From steady state kinetic studies, a Km for cyclohexanol of 8.7 mM and a Vmax of 5.7 nmol of cyclohexanone formed/min per nmol of P-450 were determined. Similarly, a Km for benzene of 105 mM and a Vmax of 22 nmol of phenol formed/min per nmol of P-450 were obtained. With intact microsomes from phenobarbital-treated rabbits, a Km for benzene of 18 mM and a Vmax of 1.7 nmol of phenol formed/min per nmol of P-450 were determined. With the use of substrate concentrations in the range of the respective Km values, superoxide dismutase, desferrioxamine and dimethyl sulfoxide had no significant effect on the P-450-catalyzed reactions. When the oxidation of benzene or cyclohexanol was examined in a model hydroxyl radical-generating system containing xanthine, xanthine oxidase, and Fe-EDTA, no dependence of the rate of oxidation on the substrate concentrations used was observed. Since the rate of hydroxyl radical generation by the model system was adjusted to be greater than the rate of product formation in the P-450 system, the lack of dependence on substrate concentration suggests that free hydroxyl radicals are not involved in the P-450-catalyzed reactions studied. The free hydroxyl radical-mediated pathway observed by other investigators evidently does not contribute significantly to product formation when these substrates are present at concentrations within the range of their respective Km values.