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Dissimilatory sulfate reduction is carried out by a heterogeneous group of bacteria and archaea that occur in environments with temperatures up to 105 degrees C. As a group together they have the capacity to metabolize a wide variety of compounds ranging from hydrogen via typical organic fermentation products to hexadecane, toluene, and several types of substituted aromatics. Without exception all sulfate reducers activate sulfate to APS; the natural electron donor(s) for the ensuing APS reductase reaction is not known. The same is true for the reduction of the product bisulfite; in addition there is still some uncertainty as to whether the pathway to sulfide is a direct six-electron reduction of bisulfite or whether it involves trithionate and thiosulfate as intermediates. The study of the degradation pathways of organic substrates by sulfate-reducing prokaryotes has led to the discovery of novel non-cyclic pathways for the oxidation of the acetyl moiety of acetyl-CoA to CO2. The most detailed knowledge is available on the metabolism of Desulfovibrio strains, both on the pathways and enzymes involved in substrate degradation and on electron transfer components and terminal reductases. Problems encountered in elucidating the flow of reducing equivalents and energy transduction are the cytoplasmic localization of the terminal reductases and uncertainties about the electron donors for the reactions catalyzed by these enzymes. New developments in the study of the metabolism of sulfate-reducing bacteria and archaea are reviewed.

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Desulfovibrio gigas NCIMB 9332 is a sulfate-reducing bacterium which is able to grow with H 2 , L-and D-lactate, C 4 -dicarboxylic acids, and ethanol as energy sources (17,35). D. gigas not only oxidizes ethanol (to acetate) but it can also store massive amounts of polyglucose (29) which under fermentative conditions can be degraded with ethanol as a major end product (26).Recently, we characterized the NAD-dependent alcohol dehydrogenase from D. gigas and showed it to be an oxygen-labile, decameric enzyme (14). From lactate-grown cells, a molybdenum iron-sulfur protein (MOP) which had 2,6-dichlorophenol-indophenol (DCPIP)-linked aldehyde dehydrogenase activity (1, 31) was purified. In extracts of cells grown on lactate or ethanol, high levels of activity of a coenzyme A-independent, benzylviologen-linked aldehyde dehydrogenase (BV-AlDH) were measured (17). Because at that time only the MOP was known as an enzyme with aldehyde dehydrogenase activity in D. gigas, it was suggested that the BV-AlDH activity was catalyzed by the MOP. Recently, we found that the addition of 0.1 M tungstate to the medium had a strongly stimulatory effect on the growth of D. gigas on ethanol. Omission of tungstate from the medium resulted in a decrease or absence of the BV-AlDH activity and an increase of the DCPIP-linked aldehyde dehydrogenase activity (13). These data and the different characteristics of the enzyme activities in cell extracts strongly suggested that the BV-AlDH and the MOP are two different enzymes.In this report, we describe the purification and characterization of the BV-AlDH and provide evidence that this enzyme belongs to the group of molybdopterin-and tungsten-containing aldehyde:acceptor oxidoreductases. MATERIALS AND METHODSOrganism, cultivation, and preparation of cell extract. D. gigas NCIMB 9332 was cultivated on ethanol, harvested, washed, and stored as described earlier (14), with the following minor modifications. The washing buffer was 50 mM potassium phosphate (pH 7.5) containing 2 mM dithiothreitol (DTT) and 1 mM dithionite. Cell extract was prepared by three successive passages through a French pressure cell operated at 106 MPa and centrifugation (20 min at 48,000 ϫ g and 4ЊC). The cell extract was then used for enzyme purification or stored at Ϫ20ЊC under N 2 until further use.Enzyme assays. Assay systems in cuvettes were made anaerobic by bubbling with N 2 for at least 3 min and were then closed with grey butyl rubber stoppers. The standard assay contained 50 mM potassium phosphate buffer (pH 7.5) and 2 mM benzylviologen in a total volume (after the following additions) of 1 ml. Then, 5 l of a freshly prepared dithionite solution (1.5 mM) and enzyme solution (usually 10 l) were added with microsyringes and the reaction was started by adding acetaldehyde to a final concentration of 1 mM; oxygen was removed from acetaldehyde stock solutions in crimp-seal bottles by replacing the atmosphere with oxygen-free nitrogen twice. Addition of dithionite was necessary to prevent a lag phase in the reaction. Ac...