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We show that the pseudoperoxidase activity of ferrylmyoglobin (MbTM) promotes oxidative degradation of doxorubicin (DOX), an anticancer anthracycline known to induce severe cardiotoxicity. MbIV, formed in vitro by reacting horse heart MbIII with H2O2, caused disappearance of the spectrum of DOX at 477 nm and appearance of UV-absorbing chromophores that indicated opening and degradation of its tetracyclic ring. Electron spray ionization mass spectrometry analyses of DOXfMbIV ultrafiltrates showed that DOX degradation resulted in formation of 3-methoxyphthalic acid, the product of oxidative modifications of its methoxy-substituted ring D. Other methoxy-substituted anthracyclines similarly released 3-methoxyphthalic acid after oxidation by MbIV, whereas demethoxy analogs released simple phthalic acid. Kinetic and stoichiometric analyses of reactions between DOX and MbIII/H2O2 or hemin/H2O2 showed that the porphyrin radical of MbIV-compound I and the iron-oxo moiety of MbIV-compound II were sequentially involved in oxidizing DOX; however, oxidation by compound I formed more 3-methoxyphthalic acid than oxidation by compound II. Sizeable amounts of 3-methoxyphthalic acid were formed in the heart of mice treated with DOX, in human myocardial biopsies exposed to DOX in vitro, and in human cardiac cytosol that oxidized DOX after activation of its endogenous myoglobin by H2O2. Importantly, H9c2 cardiomyocytes were damaged by low concentrations of DOX but could tolerate concentrations of 3-methoxyphthalic acid higher than those measured in murine or human myocardium. These results unravel a novel function for MbIV in the oxidative degradation of anthracyclines to phthalic acids and suggest that this may serve a salvage pathway against cardiotoxicity. [ABSTRACT FROM AUTHOR]