Conformational Dynamics of Na ⁺ -Pumping NADH-Quinone Oxidoreductase during Na ⁺ Translocation from AlphaFold-Facilitated Markov State Modeling

High Resolution Image Download MS PowerPoint Slide The Na + -pumping NADH–quinone oxidoreductase (Na + -NQR) is a respiratory chain enzyme found in pathogenic bacteria, including Vibrio cholerae, and is essential for energy metabolism by generating a transmembrane Na + gradient that drives ATP synthesis and flagellar motility. Because the molecular structure of Na + -NQR is unrelated to the corresponding mitochondrial H + -pumping NADH-quinone oxidoreductase (respiratory complex I), it is a promising antibiotic target. Although it has been shown that Na + pumping is mediated by an alternating-access conformational change in the NqrD/E subunits coupled to redox switching of a cofactor, the thermodynamics and kinetics of the conformational transition, including the free energy profile and the rate-limiting steps, remain unclear. Here, we construct redox-state-dependent Markov state models (MSMs) from extensive molecular dynamics (MD) trajectories in the oxidized and reduced states to quantify the conformational free energy landscapes and primary transition pathway. To accelerate conformational sampling, MD simulations are initiated from diverse NqrD/E conformations generated by AlphaFold. Our analysis clarifies how the NqrD/E conformation is regulated by the redox state and by Na + binding to achieve Na + translocation. This study provides a quantitative framework for understanding the ion-pumping mechanisms of redox-driven membrane proteins.