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 ions (Na⁺ or H⁺) across a membrane, powering ATP synthesis. Rnf is widespread in primordial organisms and the evolutionary predecessor of the Na⁺-pumping NADH-quinone oxidoreductase (Nqr). By running in reverse, Rnf uses the electrochemical ion gradient to drive ferredoxin reduction with NADH, providing low potential electrons for nitrogenases and CO₂ reductases. Yet, the molecular principles that couple the long-range electron transfer to Na⁺ translocation remain elusive. Here, we resolve key functional states along the electron transfer pathway in the Na⁺-pumping Rnf complex from Acetobacterium woodii using redox-controlled cryo-electron microscopy 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 electrostatically attracts Na⁺, 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.