A carboxylate switch point controls long-range energy transduction in respiratory Complex I

Complex I is a highly intricate membrane-bound protein complex that powers the cellular energy metabolism by a long-range ( > 300 Å) proton-coupled electron transfer (PCET) reaction. Here, we investigate the highly debated coupling mechanism of Complex I by probing the charge transfer reaction along its functionally central carboxylate pathway (E-channel). By combining biophysical and site-directed mutagenesis experiments with high-resolution (2.6-2.8 Å) cryo-electron microscopy (cryo-EM) and multiscale simulations, we identify a conserved carboxylate switch point (D79^NuoA) that mediates proton transfer by establishing a kinetic gate and couples the redox chemistry to proton pumping. We find that mutation of the identified site, as found in patients suffering from severe neurodegenerative disorders, drastically perturbs the charge transfer mechanism, and results in a 20% PCET activity. Our combined findings illustrate mechanistic principles of molecular gates underlying long-range charge transfer reactions, and show how disease mutations perturb the function of conserved switch points in energy transduction.