The mitochondrial electron transport chain (ETC) is an intricate metabolic pathway, which together with the rotary enzyme ATP synthase, is responsible for majority of ATP production in the cell. The ETC consists of four respiratory complexes (I-IV) embedded in the inner membrane of mitochondria. The respiratory complexes I, III, and IV, which are all redox-driven proton pumps, have been studied with several experimental and computational approaches . In contrast to advanced mechanistic understanding of complexes III and IV, the mechanism of the first and the largest protein complex of ETC, respiratory complex I, remains largely unclear. Respiratory complex I from mammalian mitochondria consists of over 40 individual protein subunits and several redox-active cofactors. It catalyzes the reduction of ubiquinone (Q) to ubiquinol (QH2) with two electrons and two protons. The energy released in the redox reaction drives the pumping of four protons across the inner mitochondrial membrane (Fig. ) . It is generally agreed that the proton pumping stoichiometry of complex I is four pumped protons per two electrons consumed for Q reduction .