ALKOXYL AND METHYL RADICAL FORMATION DURING CLEAVAGE OF TERT-BUTYL HYDROPEROXIDE BY A MITOCHONDRIAL MEMBRANE-BOUND, REDOX ACTIVE COPPER POOL - AN EPR STUDY
Em. Massa et C. Giulivi, ALKOXYL AND METHYL RADICAL FORMATION DURING CLEAVAGE OF TERT-BUTYL HYDROPEROXIDE BY A MITOCHONDRIAL MEMBRANE-BOUND, REDOX ACTIVE COPPER POOL - AN EPR STUDY, Free radical biology & medicine, 14(5), 1993, pp. 559-565
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.