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J. Bacteriol. doi:10.1128/JB.01017-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Molecular Microbiology & Bioenergetics, Institute of Molecular Biosciences, Johann Wolfgang Goethe University Frankfurt/Main, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany; Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-University, 35032 Marburg, Germany

^* To whom correspondence should be addressed. Email: vmueller{at}bio.uni-frankfurt.de.

	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 have addressed the nature of the hitherto unknown enzymatic activities involved in this process and their cellular localization. Cell free extract of A. woodii catalyzes H₂-dependent caffeate reduction. This reaction is strictly ATP dependent, but can be activated also by acetyl coenzyme A, indicating the formation of caffeyl-CoA prior to reduction. Two-dimensional gel electrophoresis revealed proteins present only in caffeate-grown cells. Two proteins were identified by ESI-MS/MS and the encoding genes were cloned. They 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 electron donor for cytosolic caffeate reduction. Since the hydrogenase was soluble and used ferredoxin as electron acceptor, the missing link was a ferredoxin:NAD-oxidoreductase. This activity could be determined and most interestingly was membrane-bound. Searching 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: H₂ > ferredoxin > NAD⁺ > Etf > caffeate reductase and implies that the sodium-motive step in the chain is the ferredoxin-dependent NAD⁺ reduction catalyzed by Rnf.

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