A component of the mitochondrial respiratory chain located between the sites of the antimycin and rotenone inhibition has been shown to produce considerable amounts of H202 under certain conditions [l-4]. Since this part of the respiratory chain switches from two electron transfer to one electron transfer H202 may be formed either directly by a two electron or indirectly by a one electron carrier via 0, radicals. This paper tries to indentify a free radical precursor of mitochondrial H202, which might provide insight into the stoichiometry of the primary process. The question of a free radical formation in mitochondria may also be relevant to physiopathology, since eg 0; and one product of its spontaneous dismutation, singlet oxygen, OS, are considered to be more toxic than H202 itself [S, 61. In addition, a continuous generation of 0, radicals by the respiratory chain would disclose a physiological need for the presence of superoxide dismutase (SOD) in the mitochondrial matrix recently discovered by Weisiger and Fridovich [7].

With the aid of bovine blood SOD as an indicator enzyme, we were able to demonstrate that the superoxide radical, at least under some conditions, is a precursor of H202 generated by submitochondrial particles (SMP). Some observations, however, revealed an alternative pathway of mitochondrial H20, formation apparently involving an oxygen species distinct from 0,.