Study of the redox properties of naphthazarin (5,8-dihydroxy-1,4-naphthoquinone) and its glutathionyl conjugate in biological reactions: One- and two-electron enzymatic reduction

Abstract

Naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), the basic unit of several tetracyclic antitumor antibiotics, and its glutathione conjugate were reduced by the one- and two-electron transfer flavoproteins NADPH-cytochrome P450 reductase and DT-diaphorase to their semi- and hydroquinone forms, respectively. Kinetic studies performed on purified DT-diaphorase showed the following results: K m NADPH = 68 μM, K m Quinone = 0.92 μM, and V max 1300 nmol × min −1 × μg enzyme −1. Similar studies performed on purified NADPH-cytochrome P450 reductase indicated a lower K m NADPH (10.5 μ m) and higher K m Quinone (2.3 μ m). The V max values were 20-fold lower (46 nmol × min −1 × μg enzyme −1) than those observed with DT-diaphorase. DT-diaphorase reduced the naphthazarin-glutathione conjugate with an efficiency 5-fold lower than that observed with the parent quinone. The nucleophilic addition of GSH to naphthazarin proceeded with GSH consumption at rates slower than those observed with 1,4-naphthoquinone and its monohydroxy derivative, 5-hydroxy-1,4-naphthoquinone. The initial rate of GSH consumption during these reactions did not vary whether the assay was carried out under anaerobic or aerobic conditions. Autoxidation accompanied the DT-diaphorase and NADPH-cytochrome P450 reductase catalysis of naphthazarin and its glutathionyl adduct as well as the 1,4-reductive addition of GSH to naphthazarin. Superoxide dismutase at catalytic concentrations (n m range) enhanced slightly (1.1- to 1.6-fold) the autoxidation following the enzymatic catalysis of naphthazarin. Autoxidation during the GSH reductive addition to 1,4-naphthoquinones decreased with increasing number of -OH substituents, 1,4-naphthoquinone > 5-hydroxy-1,4-naphthoquinone > 5,8-dihydroxy-1,4-naphthoquinone, thus revealing that the contribution of redox transitions other than autoxidation, e.g., cross-oxidation, to the decay of the primary product of nucleophilic addition increases with increasing number of -OH substituents. Superoxide dismutase enhanced substantially the autoxidation of glutathionyl-naphthohydroquinone adducts, thereby affecting only slightly the total GSH consumed and GSSG formed during the reaction. The present results are discussed in terms of the relative contribution of one- and two-electron transfer flavoproteins to the bioreductive activation of naphthazarin and its glutathionyl conjugate as well as the importance of autoxidation reactions in the mechanism(s) of quinone cytotoxicity.