The calcium sensor ruthenium red can act as a Fenton-type reagent

Ruthenium red (RR), an ammoniated form of tris-ruthenium(III,IV,III) oxychloride, has been widely used in the micromolar range as a strong and specific inhibitor of in vitro and in vivo Ca(2+)-mediated biochemical processes without regard for its redox properties. We show here that in the presence of tert-butyl hydroperoxide (TBHP) and an electron source, either succinate-energized rat liver mitochondria or ascorbate, RR amplifies the generation of methyl radicals. The EPR spin trapping signal of the 5,5-dimethyl-1-pyrroline-N-oxide/methyl radical (DMPO/.CH3) adduct obtained from incubations of TBHP (1.5 mM) and mitochondria (5 mg protein/ml) in an adequate medium increases upon addition of RR in a concentration-dependent fashion: sixfold at 10 microM RR. Respiring mitochondria can be replaced by ascorbate (1 mM), the characteristic EPR signal of the ascorbyl radical also being observed (aH = 0.18 mT). Spectrophotometric, cyclic voltammetric and spectroelectrochemical studies unequivocally show oxidation of RR(III,IV,III) (lambda max = 538 nm) to the ruthenium(IV,III,IV) species ("ruthenium brown," RB; lambda max = 464 nm) by TBHP, followed by its one-electron back reduction to RR by the respiratory chain or ascorbate. The calcium chelator EGTA (1 mM) strongly binds and stabilizes the RR form, slowing down its recycling by TBHP and either ascorbate or the mitochondrial electron chain. These data clearly show that Ru(III) in the RR complex can reduce TBHP via a Fenton-type reaction and thus must be considered when RR is used as a tool to study biological processes simultaneously involving Ca2+ ions and peroxides.