Structure and function of a bacterial ion (Na+)-translocating ferredoxin: NAD+-oxidoreductase (Rnf)

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The anaerobic acetogenic bacterium Acetobacterium woodii lives at the thermodynamic limit of life. During autotrophic growth, net ATP synthesis by substrate level phosphorylation is zero, but a very simple respiratory chain consisting of a membrane-bound, Na+-translocating ferredoxin:NAD oxidoreductase and a Na+-F1FO ATP synthase is hypothesized to synthesize a little ATP in addition (0.3 mol/mol acetate formed). Our own earlier studies suggested that the ferredoxin:NAD oxidoreductase is encoded by the rnf genes, a class of genes that had been suggested already in the 1980s by others to encode a respiratory enzyme with similarity to the Na+-translocating NADH:quinone oxidoreductase. The rnf genes are widespread in different phylogenetic groups of bacteria and in some archaea. However, detailed knowledge about the function and physiological role of the Rnf complex is scarce. In the last funding period, we characterized the physiological role of Rnf in A. woodii in more detail, addressed the biochemistry and bioenergetics of the activity, and determined properties of the Rnf complex and subunits thereof. Deletion analyses using a bacterium and an archaeon provided evidence that the ferredoxin:NAD oxidoreductase activity is indeed encoded by the rnf genes. We established a reliable enzymatic assay for the complex that allowed unequivocal demonstration of its Na+ dependence and enriched a Na+-Rnf complex from Thermotoga maritima, together with the Na+-ATP synthase. A very important observation was that dicyclohexylcarbodiimide (DCCD) inhibited ferredoxin-dependent NAD reduction and that this inhibition was prevented by preincubation with Na+. In addition, we succeeded in producing proteins homologously in A. woodii which will open the door for a structure-function analysis of the Rnf complex. We will follow up the data obtained during the first funding period and will pursue two major goals. First, the Na+-Rnf and Na+-F1FO ATP synthase preparation described above offers the unique opportunity to test the hypothesis that the Rnf activity is coupled to ion transport and the generation of a membrane potential that then drives ATP synthesis. To this end, Rnf and ATP synthase present in the preparation will be co-reconstituted into liposomes and analyzed biochemically and bioenergetically. Second, the Rnf complex will be characterized with a focus on the subunits and residues involved in ion binding, making use of the competetion of DCCD and Na+. DCCD labeling will be used as a tool to identify the Na+ binding site(s). We have also established recently a procedure to produce proteins in A. woodii. This will allow us to verify the importance of the DCCD-binding site for Na+ transport by mutational analyses and allow to identify other residues of the Na+ binding pocket and even additional Na+-binding pockets. In sum, we will get detailed insights into the function of a novel and so far fairly unexamined respiratory enzyme.