The Rnf Complex Is an Energy-Coupled Transhydrogenase Essential To Reversibly Link Cellular NADH and Ferredoxin Pools in the Acetogen Acetobacterium woodii

The Rnf complex is a respiratory enzyme that catalyzes the oxidation of reduced ferredoxin to the reduction of NAD⁺, and the negative free energy change of this reaction is used to generate a transmembrane ion gradient. In one class of anaerobic acetogenic bacteria, the Rnf complex is believed to be essential for energy conservation and autotrophic growth. We describe here a methodology for markerless mutagenesis in the model bacterium of this class, Acetobacterium woodii, which enabled us to delete the rnf genes and to test their in vivo role. The rnf mutant did not grow on H₂ plus CO₂, nor did it produce acetate or ATP from H₂ plus CO₂, and ferredoxin:NAD⁺ oxidoreductase activity and Na⁺ translocation were also completely lost, supporting the hypothesis that the Rnf complex is the only respiratory enzyme in this metabolism. Unexpectedly, the mutant also did not grow on low-energy substrates, such as ethanol or lactate. Oxidation of these substrates is not coupled to the reduction of ferredoxin but only of NAD⁺, and we speculated that the growth phenotype is caused by a loss of reduced ferredoxin, indispensable for biosynthesis and CO₂ reduction. The electron-bifurcating hydrogenase of A. woodii reduces ferredoxin, and indeed, the addition of H₂ to the cultures restored growth on ethanol and lactate. This is consistent with the hypothesis that endergonic reduction of ferredoxin with NADH is driven by reverse electron transport catalyzed by the Rnf complex, which renders the Rnf complex essential also for growth on low-energy substrates.IMPORTANCE Ferredoxin and NAD⁺ are key electron carriers in anaerobic bacteria, but energetically, they are not equivalent, since the redox potential of ferredoxin is lower than that of the NADH/NAD⁺ couple. We describe by mutant studies in Acetobacterium woodii that the main function of Rnf is to energetically link cellular pools of ferredoxin and NAD⁺ When ferredoxin is greater than NADH, exergonic electron flow from ferredoxin to NAD⁺ generates a chemiosmotic potential. This is essential for energy conservation during autotrophic growth. When NADH is greater than ferredoxin, Rnf works in reverse. This reaction is essential for growth on low-energy substrates to provide reduced ferredoxin, indispensable for biosynthesis and CO₂ reduction. Our studies put a new perspective on the cellular function of the membrane-bound ion-translocating Rnf complex widespread in bacteria.