Recent structures of complex I from different species obtained by X-ray crystallography 2, 3 and cryo-electron microscopy 4, 5, 6, 7 showed that FMN and a chain of seven ironвЂ“sulfur clusters reside in its peripheral arm (N/Q modules) and connect the NADH oxidation site to the ubiquinone reduction site. Yet, the molecular mechanism and regulation of energy conversion and ROS production by complex I remain poorly understood. Complex I is also a key player in signaling and pathologic mechanisms associated with reactive oxygen species (ROS) in humans 1. By linking electron transfer from NADH to ubiquinone with the translocation of four protons across the inner mitochondrial membrane, it generates a major portion of the proton motive force driving aerobic ATP synthesis and the transport of metabolites. Respiratory complex I feeds redox equivalents into oxidative phosphorylation. We conclude that movement of loop TMH1-2 ND3 located at the ubiquinone-binding pocket is required to drive proton pumping corroborating one of the central predictions of our model for the mechanism of energy conversion by complex I proposed earlier. The X-ray structure of mutant complex I indicates that the disulfide bridge immobilizes but does not displace the tip of loop TMH1-2 ND3.

Here we show that fixing loop TMH1-2 ND3 to the nearby subunit PSST via a disulfide bridge introduced by site-directed mutagenesis reversibly disengages proton pumping without impairing ubiquinone reduction, inhibitor binding or the Active/Deactive transition. We hypothesized that during energy conversion by complex I, electron transfer onto ubiquinone triggers the concerted rearrangement of three protein loops of subunits ND1, ND3, and 49-kDa thereby generating the power-stoke driving proton pumping.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the largest enzyme of the mitochondrial respiratory chain and a significant source of reactive oxygen species (ROS).

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