Kinetic and energetic investigation of mammalian mitochondrial NADH-ubiquinone oxidoreductase: Development of a multi-site model of electron transfer from complex I

Mitochondrial NADH-ubiquinone oxidoreductase (complex I) is the first coupling site for energy conservation in mitochondria. Complex I is implicated in Parkinson's disease, Alzheimer's disease, and other pathologies, possibly by reactive oxygen species (ROS) production from the enzyme. Incomplete reduction of oxygen leads to ROS like superoxide anion, hydrogen peroxide, and hydroxyl radicals, which are harmful to cells. This ROS production from isolated complex I in these diseases has not been measured and ROS contributions to these diseases are speculative. The complexity of complex I has hindered investigation of complex I ROS production, physiological electron transfer, and proton transport.; ROS production co-isolated with complex I, was dependent on the presence of ubiquinone, and was enhanced by piericidin A inhibition. Arrhenius activation energies showed different rate-limiting steps for the reduction of ubiquinone substrates, Q₁ and DB. Also, piericidin A-sensitive DB reduction was more sensitive to detergent/salt fractionations than Q₁ reduction. Kinetic analysis using the inhibitor myxothiazol also showed different kinetic profiles for Q₁ and DB. This data supports the notion of two classes of inhibitor-sensitive ubiquinone reduction sites on complex I. Single electron transfer reactions from complex I, which are initial steps in ROS production, were resolved by Arrhenius analysis of inhibitor-sensitive and -insensitive reactions. Rates of electron transfer between complexes I and III indicate that ROS production and the fidelity of electron transfer are not independent.; A stable ubisemiquinone species present in complex I was sensitive to piericidin A and had a midpoint potential of −43 mV. This species showed microwave power saturation behavior consistent with the slow-relaxing ubisemiquinone observed in submitochondrial particles (SMP). From this data, a multi-site model of substrate and oxygen reduction for complex I is presented. This model localizes ROS production to the oxidizing side of the inhibitor block in complex I, and suggests a possible role for semiubiquinone observed in isolated complex I in physiological electron transfer. This multi-site model for electron transfer assists in the understanding of the biochemistry of the physiological mechanism of electron transfer and the conditions that support ROS production that contribute to pathophysiological states associated with complex I.