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

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 ion-pumping mechanisms of redox-driven membrane proteins.