ALKOXYL AND METHYL RADICAL FORMATION DURING CLEAVAGE OF TERT-BUTYL HYDROPEROXIDE BY A MITOCHONDRIAL MEMBRANE-BOUND, REDOX ACTIVE COPPER POOL - AN EPR STUDY

The cleavage of tert-butyl hydroperoxide by submitochondrial particles yielded two distinctive radicals, alkoxyl and methyl radicals, detect ed by the electron paramagnetic resonance technique in conjunction wit h the spin trap 5,5'-dimethyl-1-pyrrolyne-N-oxide. Free radical format ion was partly sensitive to bathocuproine disulfonate and was augmente d upon supplementation of the mitochondrial membranes with copper, thu s suggesting that a redox active copper pool in mitochondrial membrane s participated actively in the cleavage of the O-O bond of tert-butyl hydroperoxide. This view was experimentally substantiated by the follo wing: first, observation of the maximal EPR signal intensity required the presence of exogenous electron donors, such as succinate or reduce d nicotinamide adenine dinucleotide (NADH). Second, copper reduction w as accomplished partly by a superoxide radical-dependent mechanism as indicated by the sensitivity of the electron paramagnetic resonance (E PR) signal to superoxide dismutase. Third, the enhancing effect of the respiratory chain inhibitors, antimycin A and rotenone, on free radic al yield assessed in the presence of superoxide dismutase pointed to t he occurrence of two potential loci in the respiratory chain involved in direct electron transfer to membrane-bound copper. one located betw een NADH and the rotenone-sensitive site and another, quantitatively l ess important, between the rotenone- and antimycin-sensitive sites. Th ese results support the notion that a redox pool of copper tightly bou nd to the mitochondrial membrane contributes significantly to the redu ctive cleavage of organic peroxides associated with free radical produ ction.