BPS2026 – Ion translocation mechanism of Na+-pumping NADH-quinone oxidoreductase from molecular dynamics simulations

The Na + -pumping NADH-ubiquinone oxidoreductase (Na + -NQR) is a respiratory enzyme of numerous marine and pathogenic bacteria such as Vibrio cholerae that utilizes NADH-mediated reduction as a driving force to generate a Na + gradient across the membrane. The molecular structure consists of six subunits (NqrA-F) and six redox cofactors such as riboflavin derivatives and iron-sulfur clusters, which has been revealed by cryo-electron microscopy. However, the pathway and mechanism of Na + translocation across the membrane remain unclear. In this study, we performed redox-switched atomistic molecular dynamics (MD) simulations and revealed the redox-state-dependent Na + translocation mechanism. First, we revealed that Na + binds to the iron-sulfur cluster buried in the transmembrane NqrD/E subunits (2Fe-2S NqrD/E ) in the reduced state. Then, a targeted MD simulation with the reduced 2Fe-2S NqrD/E and subsequent equilibration with the oxidized 2Fe-2S NqrD/E showed that the alternating-access mechanism of the transmembrane helices in NqrD/E mediates Na + translocation. Furthermore, we developed Markov state models to explore the energetics, driving force, and primary pathway of the conformational transition underlying Na + translocation by Na + -NQR. The Markov state models indicated that Na + -binding to the reduced 2Fe-2S NqrD/E stabilizes the occluded state, which drives the conformational change in NqrD/E. The transition path analysis using the reactive fluxes suggested that the conformational transition from the inward-open to the outward-open state is coupled with Na + translocation, and the rate-limiting step is a transition from the occluded to the outward-open state.