Dissection of the Caffeate Respiratory Chain in the Acetogen Acetobacterium woodii : Identification of an Rnf-Type NADH Dehydrogenase as a Potential Coupling Site

Abstract

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 ␣ (EtfA) and ␤ (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 3 ferredoxin 3 NAD ؉ 3 Etf 3 caffeyl-CoA reductase. They also imply that the sodium motive step in the chain is the ferredoxin-dependent NAD ؉ reduction catalyzed by Rnf.

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

“…There was even less stimulation when the reducing agent Ti 3ϩ was used in the absence of ferredoxin (activity increased from 35 to 50 milliunits/mg). These data confirm our data from a previous study in which we could not unequivocally demonstrate a Na ϩ dependence of NAD ϩ reduction driven by Ti 3ϩ -reduced ferredoxin (6). In addition, they also demonstrate a ferredoxin-independent entry port(s) into the electron transport chain for electrons coming from Ti 3ϩ .…”

“…Preparation of Membranes-Membranes of fructose-grown cells were prepared as described previously (6). The cell-free extract was separated into cytoplasmic and membrane fractions by ultracentrifugation at 150,000 ϫ g for 2 h at 4°C.…”

“…In the assay that was used previously (6,11), these were provided by Ti 3ϩ (E 0 Ј ϳ Ϫ480 mV) (15). However, Ti 3ϩ is a very strong and unspecific reducing agent.…”

“…This is in contrast to other Na ϩ -translocating enzymes such as decarboxylases (26, 27), methyltransferases (28, 29), and ATP synthases (25, 30, 31) or flagellar motors (4,32), in which the enzymatic activity/rotation was clearly Na ϩ -dependent. The ferredoxin:NAD ϩ oxidoreductase is usually measured using ferredoxin that has been reduced by Ti 3ϩ (6,14,19,33). This assay has a number of inherent problems such as a low concentration of "electrons," and the assay does not reliably require ferredoxin, indicating that Ti 3ϩ -derived electrons enter the enzyme at different sites and direct non-enzymatic electron flow from Ti 3ϩ to NAD ϩ at pH values above 7.…”

“…Acetogenesis as carried out by resting cells is coupled to the generation of a transmembrane electrochemical Na ϩ gradient across the cytoplasmic membrane that is the driving force for the above-mentioned endergonic membrane processes. The nature of the enzyme(s) that generates ⌬ Na ϩ has been obscure for decades, but we recently demonstrated a ferredoxin:NAD ϩ oxidoreductase activity at the cytoplasmic membrane of A. woodii (6). This enzyme is part of the electron transport chain leading from, for example, hydrogen to carbon dioxide or caffeate as electron acceptor.…”