NADH:ubiquinone oxidoreductase (complex I) from bovine heart mitochondria is a complicated, energy-transducing, membrane-bound
enzyme that contains 45 different subunits, a non-covalently bound
flavin mononucleotide, and eight
iron-
sulfur clusters. The mechanisms of
NADH oxidation and intramolecular electron transfer by complex I are gradually being defined, but the mechanism linking
ubiquinone reduction to
proton translocation remains unknown. Studies of
ubiquinone reduction by isolated complex I are problematic because the extremely hydrophobic natural substrate, ubiquinone-10, must be substituted with a relatively hydrophilic analogue (such as
ubiquinone-1). Hydrophilic ubiquinones are reduced by an additional, non-energy-transducing pathway (which is insensitive to inhibitors such as
rotenone and
piericidin A). Here, we show that inhibitor-insensitive
ubiquinone reduction occurs by a ping-pong type mechanism, catalyzed by the
flavin mononucleotide cofactor in the active site for
NADH oxidation. Moreover, semiquinones produced at the
flavin site initiate redox cycling reactions with molecular
oxygen, producing
superoxide radicals and
hydrogen peroxide. The
ubiquinone reactant is regenerated, so the
NADH:Q reaction becomes superstoichiometric.
Idebenone, an artificial
ubiquinone showing promise in the treatment of
Friedreich's Ataxia, reacts at the
flavin site. The factors which determine the balance of reactivity between the two sites of
ubiquinone reduction (the energy-transducing site and the
flavin site) and the implications for mechanistic studies of
ubiquinone reduction by complex I are discussed. Finally, the possibility that the
flavin site in complex I catalyzes redox cycling reactions with a wide range of compounds, some of which are important in pharmacology and toxicology, is discussed.