Fig. 4: Redox-driven Na⁺ translocation mechanism in the Rnf complex.

a Schematic representation of the redox-driven Na⁺ translocation mechanism. i: Fd transfers an electron via a chain of FeS cluster in RnfB to the terminal B8 cluster. ii: Reduction of B8 induces a conformational change in the B8-domain, which approaches RnfA/E on the cytosolic side. The conformational changes result in a shorter distance between B8 and AE1, enabling the electron transfer. iii: Reduction of AE1 increases the Na⁺ binding affinity in RnfA/E that leads to Na⁺ uptake from the cytosol. iv: After Na⁺ binding next to AE1, RnfA/E undergoes an inward/outward transition that, in turn, moves RnfG closer to RnfA/E. v: Electron transfer from AE1 to FMN^G lowers the binding affinity for Na⁺, and results in the Na⁺ release to the extracellular side. The electron is transferred from FMN^G to the RnfC FeS via the flavins on RnfD, in a process that couples to the relaxation of RnfA/E to its inward-open state. The reduction of C2 increases the affinity of RnfC for NAD⁺, which binds next to the FMN^C. vi-ix: similar to previous steps, another electron transfer from Fd couples to the translocation of a second Na⁺. x: Two-electron reduction yields FMNH^-, which reduces NAD⁺ to NADH via a hydride transfer, initiating a new pumping cycle. b Schematic free energy profile of the proposed Na⁺ pumping mechanism (see Supplementary Methods, and Supplementary Table 8). c Kinetic simulations of the redox-driven Na⁺ translocation process with a 0 mV (solid line) and 180 mV (dotted line) SMF. d 2D free energy profiles of the inward-outward transition coupled to the Na⁺ translocation with AE1 reduced (left) or oxidised (right). RC₁—distance between TM4 of RnfA and RnfE; RC₂—distance of Na⁺ and the centre of mass of RnfA/E, projected onto the Z-axis (see Supplementary Figs. 18–20 for details). e Structures along the free energy profile with AE1 reduced: RnfA/E (top), closeup of the Na⁺, AE1, and residues interacting with Na⁺ (bottom). Left: inward state; middle: transition state (TS); right: outward state.