The Calcium Sensor Ruthenium Red Can Act as a Fenton-Type Reagent

Ruthenium red (RR), an ammoniated form oftris-rutheniumIII,IV,IIIoxychloride, has been widely used in the micromolar range as a strong and specific inhibitor ofin vitroandin vivoCa2+-mediated biochemical processes without regard for its redox properties. We show here that in the presence oftert-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 μMRR. 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 RRIII,IV,III(λmax= 538 nm) to the rutheniumIV,III,IVspecies (“ruthenium brown,” RB; λmax= 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 RuIIIin 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.