Molecular principles of redox-coupled sodium pumping of the ancient Rnf machinery

The Rnf complex is the primary respiratory enzyme of several anaerobic prokaryotes that transfers electrons from ferredoxin to NAD ⁺ and pumps sodium ions (Na ⁺ ) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na ⁺ -pumping NADH-quinone oxidoreductase (Nqr) ¹ . By running in reverse, Rnf reduces ferredoxin with NADH as reductant at the expense of the transmembrane electrochemical ion gradient and provides low potential electrons for nitrogenases as well as CO ₂ reductases. Yet, the molecular principles that couple the long-range electron transfer to the Na ⁺ translocation across the membrane remain elusive. Here we resolve key functional states along the electron transfer pathway using redox-controlled cryo-electron microscopy (cryo-EM) that, in combination with biochemical functional assays and atomistic molecular simulations, provide key insight into the redox-driven Na ⁺ pumping mechanism. We show that the reduction of the unique membrane-embedded [2Fe2S] cluster in the vestibule between the RnfA/E subunits electrostatically attracts the sodium ions, and in turn, triggers an inward / outward transition with alternating membrane access driving the Na ⁺ pump and the reduction of NAD ⁺ . Our study unveils an ancient mechanism for redox-driven ion pumping, and provides key understanding of the fundamental principles governing energy conversion in biological systems.