Bacterial Na+-translocating ferredoxin:NAD+ oxidoreductase.

The anaerobic acetogenic bacterium Acetobacterium woodii carries out a unique type of Na(+)-motive, anaerobic respiration with caffeate as electron acceptor, termed "caffeate respiration." Central, and so far the only identified membrane-bound reaction in this respiration pathway, is a ferredoxin:NAD(+) oxidoreductase (Fno) activity. Here we show that inverted membrane vesicles of A. woodii couple electron transfer from reduced ferredoxin to NAD(+) with the transport of Na(+) from the outside into the lumen of the vesicles. Na(+) transport was electrogenic, and accumulation was inhibited by sodium ionophores but not protonophores, demonstrating a direct coupling of Fno activity to Na(+) transport. Results from inhibitor studies are consistent with the hypothesis that Fno activity coupled to Na(+) translocation is catalyzed by the Rnf complex, a membrane-bound, iron-sulfur and flavin-containing electron transport complex encoded by many bacterial and some archaeal genomes. Fno is a unique type of primary Na(+) pump and represents an early evolutionary mechanism of energy conservation that expands the redox range known to support life. In addition, it explains the lifestyle of many anaerobic bacteria and gives a mechanistic explanation for the enigma of the energetic driving force for the endergonic reduction of ferredoxin with NADH plus H(+) as reductant in a number of aerobic bacteria.

A Na+-translocating pyrophosphatase in the acetogenic bacterium Acetobacterium woodii.

The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii.

Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes.

Microbes have a fascinating repertoire of bioenergetic enzymes and a huge variety of electron transport chains to cope with very different environmental conditions, such as different oxygen concentrations, different electron acceptors, pH and salinity. However, all these electron transport chains cover the redox span from NADH + H(+) as the most negative donor to oxygen/H(2)O as the most positive acceptor or increments thereof. The redox range more negative than -320 mV has been largely ignored. Here, we have summarized the recent data that unraveled a novel ion-motive electron transport chain, the Rnf complex, that energetically couples the cellular ferredoxin to the pyridine nucleotide pool. The energetics of the complex and its biochemistry, as well as its evolution and cellular function in different microbes, is discussed.

Mass spectrometry defines the stoichiometry of ribosomal stalk complexes across the phylogenetic tree.

The ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. It has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. Here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. Specifically we targeted ribosomes with different optimal growth temperatures. Our results showed that for the mesophilic bacterial ribosomes we investigated the stalk complexes are exclusively pentameric or entirely heptameric in the case of thermophilic bacteria, whereas we observed only pentameric stalk complexes in eukaryotic species. We also found the surprising result that for mesophilic archaea, Methanococcus vannielii, Methanococcus maripaludis, and Methanosarcina barkeri, both pentameric and heptameric stoichiometries are present simultaneously within a population of ribosomes. Moreover the ratio of pentameric to heptameric stalk complexes changed during the course of cell growth. We consider these differences in stoichiometry within ribosomal stalk complexes in the context of convergent evolution.

The ins and outs of Na(+) bioenergetics in Acetobacterium woodii.

The acetogenic bacterium Acetobacterium woodii uses a transmembrane electrochemical sodium ion potential for bioenergetic reactions. A primary sodium ion potential is established during carbonate (acetogenesis) as well as caffeate respiration. The electrogenic Na(+) pump connected to the Wood-Ljungdahl pathway (acetogenesis) still remains to be identified. The pathway of caffeate reduction with hydrogen as electron donor was investigated and the only membrane-bound activity was found to be a ferredoxin-dependent NAD(+) reduction. This exergonic electron transfer reaction may be catalyzed by the membrane-bound Rnf complex that was discovered recently and is suggested to couple exergonic electron transfer from ferredoxin to NAD(+) to the vectorial transport of Na(+) across the cytoplasmic membrane. Rnf may also be involved in acetogenesis. The electrochemical sodium ion potential thus generated is used to drive endergonic reactions such as flagellar rotation and ATP synthesis. The ATP synthase is a member of the F(1)F(O) class of enzymes but has an unusual and exceptional feature. Its membrane-embedded rotor is a hybrid made of F(O) and V(O)-like subunits in a stoichiometry of 9:1. This stoichiometry is apparently not variable with the growth conditions. The structure and function of the Rnf complex and the Na(+) F(1)F(O) ATP synthase as key elements of the Na(+) cycle in A. woodii are discussed.

Genetic, immunological and biochemical evidence for a Rnf complex in the acetogen Acetobacterium woodii.

Acetogenic bacteria grow by the oxidation of various substrates coupled to the reduction of carbon dioxide (acetogenesis) or other electron acceptors but the mechanisms of energy conservation are still enigmatic. Here, we report the presence of a rnf gene cluster rnfCDGEAB in Acetobacterium woodii that is speculated to encode a novel, energy-conserving ferredoxin:NAD(+)-oxidoreductase complex composed of at least six different subunits. Transcriptional analysis revealed that the genes constitute an operon. RnfC and RnfG were heterologously produced and antibodies were generated. Western blot analyses demonstrated that these subunits were produced and are associated with the cytoplasmic membrane. The subunits were present in cells respiring with either carbon dioxide or caffeate. A preparation with NADH dehydrogenase activity was obtained from detergent solubilized membranes that contained RnfC and RnfG.

Discovery of a ferredoxin:NAD+-oxidoreductase (Rnf) in Acetobacterium woodii: a novel potential coupling site in acetogens.

Acetogens use the Wood-Ljungdahl pathway for reduction of carbon dioxide to acetate. This pathway not only allows reoxidation of reducing equivalents during heterotrophic growth but also supports chemolithoautotrophic growth on H(2) + CO(2). The latter argues for this pathway being a source for net energy conservation, but the mechanism involved remains unknown. In addition to CO(2), acetogens can use alternative electron acceptors, such as nitrate or caffeate. Caffeate respiration in the model acetogen Acetobacterium woodii is coupled to energy conservation via a chemiosmotic mechanism, with Na(+) as coupling ion. The pathway and its bioenergetics were solved in some detail very recently. This review focuses on the regulation of caffeate respiration, describes the enyzmes involved, summarizes the evidence for a potential Na(+)-translocating ferredoxin:NAD(+)-oxidoreductase (Rnf complex) as a new coupling site, and hypothesizes on the role of this Rnf complex in the Wood-Ljungdahl pathway.

Dissection of the caffeate respiratory chain in the acetogen Acetobacterium woodii: identification of an Rnf-type NADH dehydrogenase as a potential coupling site.

The anaerobic acetogenic bacterium Acetobacterium woodii couples caffeate reduction with electrons derived from hydrogen to the synthesis of ATP by a chemiosmotic mechanism with sodium ions as coupling ions, a process referred to as caffeate respiration. We addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell extract of A. woodii catalyzes H(2)-dependent caffeate reduction. This reaction is strictly ATP dependent but can be activated also by acetyl coenzyme A (CoA), indicating that there is formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by electrospray ionization-mass spectrometry/mass spectrometry, and the encoding genes were cloned. These proteins are very similar to subunits alpha (EtfA) and beta (EtfB) of electron transfer flavoproteins present in various anaerobic bacteria. Western blot analysis demonstrated that they are induced by caffeate and localized in the cytoplasm. Etf proteins are known electron carriers that shuttle electrons from NADH to different acceptors. Indeed, NADH was used as an electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as an electron acceptor, the missing link was a ferredoxin:NAD(+) oxidoreductase. This activity could be determined and, interestingly, was membrane bound. A search for genes that could encode this activity revealed DNA fragments encoding subunits C and D of a membrane-bound Rnf-type NADH dehydrogenase that is a potential Na(+) pump. These data suggest the following electron transport chain: H(2) --> ferredoxin --> NAD(+) --> Etf --> caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD(+) reduction catalyzed by Rnf.