Disease-mutations perturb proton transfer reactions in respiratory complex I.

Respiratory Complex I powers oxidative phosphorylation by a long-range proton-coupled electron transfer (PCET) reaction, with mutations linked to more than half of all human mitochondrial disorders. Yet, the molecular principles underlying the functional impairment remain difficult to test, as most mutations impede both the proton pumping and oxidoreductase activities due to the tightly coupled PCET process. Here, we probe how key disease mutations in the terminal ND5 subunit (NuoL/Nqo12), linked to the development of Leigh's syndrome (LS) and LHON/MELAS (F124L, M252T, D393N), affect the proton transport activity within the dissected antiporter module Nqo12. All constructs result in fully folded antiporter modules, with the introduced substitutions showing enhanced proton conduction rates across the proteoliposome membranes relative to the wild type module. Our molecular dynamics simulations reveal that the mutations perturb the internal water network and ion-pair dynamics that are central for the long-range PCET activity in Complex I. Taken together, we suggest that the mitochondrial disease mutations alter the redox-driven proton pumping activity of Complex I by perturbing the function of local proton gates, and result in an uncontrolled proton translocation across the antiporter module. The molecular consequences of disease mutations are discussed in the context of the proposed pumping mechanism.