Complex I plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. The mechanism of this highly efficient enzyme is currently unknown. Mitochondrial complex I is a major source of reactive oxygen species, which may be one of the causes of aging. Dysfunction of complex I is implicated in many human neurodegenerative diseases. We have determined several x-ray structures of the oxidized and reduced hydrophilic domain of complex I from Thermus thermophilus at up to 3.1 Å resolution. The structures reveal the mode of interaction of complex I with NADH, explaining known kinetic data and providing implications for the mechanism of reactive oxygen species production at the flavin site of complex I. Bound metals were identified in the channel at the interface with the frataxinlike subunit Nqo15, indicating possible iron-binding sites. Conformational changes upon reduction of the complex involve adjustments in the nucleotide-binding pocket, as well as small but significant shifts of several ␣-helices at the interface with the membrane domain. These shifts are likely to be driven by the reduction of nearby iron-sulfur clusters N2 and N6a/b. Cluster N2 is the electron donor to quinone and is coordinated by unique motif involving two consecutive (tandem) cysteines. An unprecedented "on/off switch" (disconnection) of coordinating bonds between the tandem cysteines and this cluster was observed upon reduction. Comparison of the structures suggests a novel mechanism of coupling between electron transfer and proton translocation, combining conformational changes and protonation/deprotonation of tandem cysteines.Complex I (NADH:ubiquinone oxidoreductase, EC 1.6.5.3) is the first enzyme of the mitochondrial and bacterial respiratory chains. It catalyzes the transfer of two electrons from NADH to quinone, coupled to the translocation of approximately four protons across the membrane, contributing to the proton-motive force required for the synthesis of ATP (1, 2). The mitochondrial enzyme consists of 45 subunits (3) with a combined mass of ϳ980 kDa. The prokaryotic enzyme is simpler, consisting of ϳ14 subunits conserved from bacteria to humans, and has a total mass of ϳ550 kDa (2). The mitochondrial and bacterial enzymes contain equivalent redox components and have a similar L-shaped structure, with the hydrophobic arm embedded in the membrane and the hydrophilic peripheral arm protruding into the mitochondrial matrix or the bacterial cytoplasm (2, 4). Thus, the bacterial enzyme represents a "minimal" model of complex I. Because of the central role of complex I in respiration, mutations in individual subunits can lead to many human neurodegenerative diseases (5). Complex I, along with complex III (bc 1 ), has been suggested to be a major source of reactive oxygen species (ROS) 2 in mitochondria, which can damage mitochondrial DNA and may be one of the causes of aging (6). Parkinson disease, at least in its sporadic form (which represents ϳ95% of cases), may be ...