The Ferredoxin:NAD+ Oxidoreductase (Rnf) from the Acetogen Acetobacterium woodii Requires Na+ and Is Reversibly Coupled to the Membrane Potential

The anaerobic acetogenic bacterium Acetobacterium woodii has a novel Na⁺-translocating electron transport chain that couples electron transfer from reduced ferredoxin to NAD⁺ with the generation of a primary electrochemical Na⁺ potential across its cytoplasmic membrane. In previous assays in which Ti³⁺ was used to reduce ferredoxin, Na⁺ transport was observed, but not a Na⁺ dependence of the electron transfer reaction. Here, we describe a new biological reduction system for ferredoxin in which ferredoxin is reduced with CO, catalyzed by the purified acetyl-CoA synthase/CO dehydrogenase from A. woodii. Using CO-reduced ferredoxin, NAD⁺ reduction was highly specific and strictly dependent on ferredoxin and occurred at a rate of 50 milliunits/mg of protein. Most important, this assay revealed for the first time a strict Na⁺ dependence of this electron transfer reaction. The K_m was 0.2 mm. Na⁺ could be partly substituted by Li⁺. Na⁺ dependence was observed at neutral and acidic pH values, indicating the exclusive use of Na⁺ as a coupling ion. Electron transport from reduced ferredoxin to NAD⁺ was coupled to electrogenic Na⁺ transport, indicating the generation of Δ$\tilde{\mu }$_Na⁺. Vice versa, endergonic ferredoxin reduction with NADH as reductant was possible, but only in the presence of Δ$\tilde{\mu }$_Na⁺, and was accompanied by Na⁺ efflux out of the vesicles. This is consistent with the hypothesis that Rnf also catalyzes ferredoxin reduction at the expense of an electrochemical Na⁺ gradient. The physiological significance of this finding is discussed.