Summary In this work we identified the gene for the tetrathionate‐forming thiosulfate dehydrogenase (TsdA) from the purple sulfur bacterium Allochromatium vinosum by sequence analysis and reverse genetics. The recombinant protein produced in Escherichia coli is a periplasmic, monomeric 25.8 kDa dihaem cytochrome c with an enzyme activity optimum at pH 4. UV‐visible and electron paramagnetic resonance spectroscopy indicate methionine (strictly conserved M222 or M236) and cysteine (C123) as probable sixth distal axial ligands of the two haem irons in TsdA. These results place TsdA in the group of c‐type cytochromes with an unusual axial histidine‐cysteine coordination of the haem iron. These proteins appear to play a pivotal role in sulfur‐based energy metabolism. Exchange of C123 to glycine rendered thiosulfate dehydrogenase inactive, proving the importance of this residue for catalysis. TsdA homologues are present in α‐, β‐, δ‐, γ‐ and ε‐Proteobacteria. Three of these were produced in E. coli and exhibited the expected enzymatic activity. The widespread occurrence of tsdA agrees with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophs that use thiosulfate as a supplemental but not as the sole energy source.

Summary Tetrathionate (S4O6 2-) is used by some bacteria as an electron acceptor and can be produced in the vertebrate intestinal mucosa from the oxidation of thiosulphate (S2O3 2-) by reactive oxygen species during inflammation. Surprisingly, growth of the microaerophilic mucosal pathogen Campylobacter jejuni under oxygen-limited conditions was stimulated by tetrathionate, although it does not possess any known type of tetrathionate reductase. Here, we identify a dihaem cytochrome c (C8j_0815; TsdA) as the enzyme responsible. Kinetic studies with purified recombinant C. jejuni TsdA showed it to be a bifunctional tetrathionate reductase/thiosulphate dehydrogenase with a high affinity for tetrathionate. A tsdA null mutant still slowly reduced, but could not grow on, tetrathionate under oxygen limitation, lacked thiosulphate-dependent respiration and failed to convert thiosulphate to tetrathionate microaerobically. A TsdA paralogue (C8j_0040), lacking the unusual His-Cys haem ligation of TsdA, had low thiosulphate dehydrogenase and tetrathionate reductase activities. Our data highlight a hitherto unrecognized capacity of C. jejuni to use tetrathionate and thiosulphate in its energy metabolism, which may promote growth in the host. Moreover, as TsdA represents a new class of tetrathionate reductase that is widely distributed among bacteria, we predict that energy conserving tetrathionate respiration is far more common than currently appreciated.

Edited by Ruma BanerjeeThe enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from ؊300 to ؉150 mV, TsdB is redox active between ؊100 and ؉300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys 330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (E m ؉350 mV). High potential ironsulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.

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