Thiosulfate dehydrogenase: a widespread unusual acidophilic c‐type cytochrome

Abstract

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.

Thiosulfate Dehydrogenase (TsdA) from Allochromatium vinosum

“…Thiosulfate dehydrogenases (TsdAs) 5 catalyze the reversible formation of a sulfur-sulfur bond between the sulfane atoms of two thiosulfate molecules, yielding tetrathionate and releasing two electrons (see Reaction 1) (1). TsdA homologues are widespread among bacteria, agreeing with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophic bacteria (1,2).…”

“…TsdA homologues are widespread among bacteria, agreeing with reports of tetrathionate formation not only by specialized sulfur oxidizers but also by many chemoorganoheterotrophic bacteria (1,2). In the purple sulfur bacterium Allochromatium vinosum, a ␥-proteobacterium and member of the Chromatiaceae family (3), thiosulfate is oxidized by two different pathways (4).…”

“…Construction of Expression Plasmids and Site-directed Mutagenesis-The A. vinosum tsdA gene coding for the mature protein without the signal peptide was amplified and cloned as described earlier (1). Point mutations were introduced into tsdA by overlap extension (31) using standard PCR with Pfu DNA polymerase (Thermo Scientific) and pPRIBAAvtsdA (1) as the template.…”

“…Point mutations were introduced into tsdA by overlap extension (31) using standard PCR with Pfu DNA polymerase (Thermo Scientific) and pPRIBAAvtsdA (1) as the template. For the TsdA-K208G exchange, two fragments were amplified with the following primers: for the first fragment, TsdA_XbaIf and LysGly_rev; for the second fragment, LysGlyfw and IBA1hind_rev.…”

“…Electronic paramagnetic resonance (EPR) spectroscopy indicated cysteine and methionine as sixth distal axial ligands of the two heme irons. Indeed, Cys 96 was proven to be essential for catalytic activity (1). At pH 4.0, AvTsdA exhibits high specific activity (28,600 units mg Ϫ1 ) with ferricyanide as artificial electron acceptor.…”