ROS (reactive oxygen species) are considered to be a major cause of cellular oxidative stress, linked to neuromuscular diseases and aging. Complex I (NADH:ubiquinone oxidoreductase) is one of the main contributors to superoxide production by mitochondria, and knowledge of its mechanism of O2 reduction is required for the formulation of causative connections between complex I defects and pathological effects. There is evidence for two distinct (but not mutually exclusive) sites of O2 reduction by complex I. Studies of the isolated enzyme largely support the participation of the reduced flavin mononucleotide in the active site for NADH oxidation, and this mechanism is supported in mitochondria by correlations between the NAD(P)+ potential and O2 reduction. In addition, studies of intact mitochondria or submitochondrial particles have suggested a mechanism involving the quinone-binding site, supported by observations during reverse electron transport and the use of 'Q-site' inhibitors. Here, we discuss extant data and models for O2 reduction by complex I. We compare results from the isolated enzyme with results from intact mitochondria, aiming to identify similarities and differences between them and progress towards combining them to form a single, unified picture.

NADH:ubiquinone oxidoreductase (complex I) is a major source of reactive oxygen species in mitochondria and a contributor to cellular oxidative stress. In isolated complex I the reduced flavin is known to react with molecular oxygen to form predominantly superoxide, but studies using intact mitochondria contend that superoxide may result from a semiquinone species that responds to the proton-motive force (Δp) also. Here, we use bovine heart submitochondrial particles to show that a single mechanism describes superoxide production by complex I under all conditions (during both NADH oxidation and reverse electron transfer). NADH-induced superoxide production is inhibited by complex I flavin-site inhibitors but not by inhibitors of ubiquinone reduction, and it is independent of Δp. Reverse electron transfer (RET) through complex I in submitochondrial particles, driven by succinate oxidation and the Δp created by ATP hydrolysis, reduces the flavin, leading to NAD+ and O2 reduction. RET-induced superoxide production is inhibited by both flavin-site and ubiquinone-reduction inhibitors. The potential dependence of NADH-induced superoxide production (set by the NAD+ potential) matches that of RET-induced superoxide production (set by the succinate potential and Δp), and they both match the potential dependence of the flavin. Therefore, both NADH- and RET-induced superoxide are produced by the flavin, according to the same molecular mechanism. The unified mechanism describes how reactive oxygen species production by complex I responds to changes in cellular conditions. It establishes a route to understanding causative connections between the enzyme and its pathological effects and to developing rational strategies for addressing them.