The reaction cycle of the reconstituted Sox enzyme system of Paracoccus pantotrophus requires the periplasmic proteins SoxYZ, SoxXA, SoxB, and SoxCD. The heme enzyme SoxXA covalently binds the sulfur substrate to the thiol of the single cysteine residue of SoxY located at its carboxy-terminal end. Bound sulfur is then oxidized to sulfate by a series of reactions. These involve sulfur dehydrogenase SoxCD which oxidizes the protein-bound sulfane sulfur to sulfone in a unique six-electron transfer. Bound sulfone is then hydrolyzed off by the sulfate thiohydrolase SoxB to regenerate SoxYZ. The flavoprotein SoxF enhances the rate of sulfur oxidation in vivo as evident from mutant analysis and we have specified its action in vitro. SoxYZ unlike the other Sox proteins is inactivated upon reduction. When the Sox system is reconstituted with inactivated SoxYZ, the thiosulfate-oxidizing activity is drastically decreased. SoxF reverses this inactivation and may mediate a conformational change of SoxYZ possibly by a transient interprotein disulfide. The membrane protein SoxV and the thioredoxin SoxS are essential for chemotrophic growth as evident from homogenote mutants defective in these proteins. Evidence is presented that both proteins transfer reductant from the cytoplasm to the periplasm and that SoxYZ is the final target of this transfer to balance the redox state of the Sox enzyme system or reduce a SoxY .. Y interprotein disulfide.

The significance of the soxS gene product on chemotrophic sulfur oxidation of Paracoccus pantotrophus was investigated. The thioredoxin SoxS was purified, and the N-terminal amino acid sequence identified SoxS as the soxS gene product. The wild-type formed thiosulfate-oxidizing activity and Sox proteins during mixotrophic growth with succinate plus thiosulfate, while there was no activity, and only traces of Sox proteins, under heterotrophic conditions. The homogenote mutant strain GBVS is unable to express the soxSR genes, of which soxR encodes a transcriptional regulator. Strain GBVS cultivated mixotrophically showed about 22 % of the specific thiosulfate-dependent O 2 uptake rate of the wild-type, and when cultivated heterotrophically it produced 35 % activity. However, under both mixotrophic and heterotrophic conditions, strain GBVS formed Sox proteins essential for sulfur oxidation in vitro at the same high level as the wild-type produced them during mixotrophic growth. Genetic complementation of strain GBVS with soxS restored the activity upon mixotrophic and heterotrophic growth. Chemical complementation by reductants such as L-cysteine, DTT and tris(2-carboxyethyl)phosphine also restored the activity of strain GBVS in the presence of chloramphenicol, which is an inhibitor of de novo protein synthesis. The data demonstrate that SoxS plays a key role in activation of the Sox enzyme system, and this suggests that SoxS is part of a novel type of redox control in P. pantotrophus.

Paracoccus pantotrophus GB17 requires thiosulfate for induction of the sulfur-oxidizing (Sox) enzyme system. The soxRS genes are divergently oriented to the soxVWXYZA-H genes. soxR predicts a transcriptional regulator of the ArsR family and soxS a periplasmic thioredoxin. The homogenote mutant GBVS carrying a disruption of soxS by the V-kanamycin-resistance-encoding interposon expressed a low thiosulfate-oxidizing activity under heterotrophic and mixotrophic growth conditions. This activity was repressed by complementation with soxR, suggesting that SoxR acts as a repressor and SoxS is essential for full expression. Sequence analysis uncovered operator characteristics in the intergenic regions soxS-soxV and soxW-soxX. In each region a transcription start site was identified by primer extension analysis. Both regions were cloned into the vector pRI1 and transferred to P. pantotrophus. Strains harbouring pRI1 with soxS-soxV or soxW-soxX expressed the sox genes under heterotrophic conditions at a low rate, indicating repressor titration. Sequence analysis of SoxR suggested a helix-turn-helix (HTH) motif at position 87-108 and uncovered an invariant Cys-80 and a cysteine residue at the C-terminus. SoxR was overproduced in Escherichia coli with an N-terminal His 6 -tag and purified to near homogeneity. Electrophoretic gel mobility shift assays with SoxR retarded the soxS-soxV region as a single band while the soxW-soxX region revealed at least two protein-DNA complexes. These data demonstrated binding of SoxR to the relevant DNA. This is believed to be the first report of regulation of chemotrophic sulfur oxidation at the molecular level.

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