Significance The electron transport chain of mitochondria is initiated by the respiratory complex I that converts chemical energy into a proton motive force to power synthesis of adenosine triphosphate. On a chemical level, complex I catalyzes elementary electron and proton transfer processes that couple across large molecular distances of >300 Å. However, under low oxygen concentrations, the respiratory chain operates in reverse mode and produces harmful reactive oxygen species. To avoid cell damage, the mitochondrial complex I transitions into a deactive state that inhibits turnover by molecular principles that remain elusive. By combining large-scale molecular simulations with cryo-electron microscopy data, we show here that complex I deactivation blocks the communication between proton pumping and redox modules by conformational and hydration changes.