Significance Complex I is the primary energy-converting enzyme of aerobic respiratory chains. By reducing quinone to quinol, this gigantic enzyme pumps protons across its membrane domain, which in turn powers ATP synthesis and active transport. Despite the recently resolved molecular structures of complex I, the quinone dynamics and its coupling to the pumping function remains unclear. Here we show by large-scale molecular simulations that the quinone reduction leads to ejection of the quinol molecule from the active site into a second binding site near the proton-pumping membrane domain of complex I. The identified region has been linked with human mitochondrial disorders. Our work suggests that the quinone dynamics provides a key coupling element in complex I.