The ability to perform the very simple oxidation of two molecules of thiosulphate to tetrathionate is wide spread among prokaryotes. Despite the widespread occurrence of tetrathionate formation, and its well-documented significance within the sulphur cycle, little is known about the enzymes catalysing the oxidative condensation of two thiosulphate anions. To fill this gap, the thiosulphate dehydrogenase (TsdA), enzyme from Allochromatium vinosum, was recombinantly expressed, purified and kinetic and spectroscopically characterized [1]. Moreover, we solved the crystal structure of the enzyme by Single Anomalous Dispersion (SAD) method using the Fe-haem anomalous signal. We have further obtained X-ray structures of TsdA in several redox states.
The protein crystallized in space group C2 with PEG 3350 as precipitant and one molecule in the asymmetric unit [2]. Initial crystallization trials rendered multiple, urchin-like crystals with no diffraction ability. Using iodide as an additive worked as a “silver bullet” allowing to obtain single crystals that diffract to 1.4 Å resolution. TsdA contains two typical class I c-type cytochrome domains with two hemes axially coordinated by His53/Cys96 and His164/Lys208. The X-ray structure showed an all-alpha structure with structural similarities to the Rhodovulum sulfidophilum’s SoxAX (PDB code 2OZ1), and the low-redox-potential cytochrom c6 from Hizikia fusiformis (PDB code 2ZBO).
Interestingly, reduction of the enzyme causes a ligand switch from Lys208 to Met209 in heme 2. TsdALys208Asn or Lys208Gly variants exhibit similar substrate affinities as the wildtype protein but much lower specific activities pointing at this heme as the electron exit point. Cys96 is essential for catalysis. Overall, our kinetic, spectroscopic and structural data lead us to propose a mechanism where two thiosulfate molecules enter the active site, inducing a movement of the Sγ of Cys96 out of the iron coordination sphere; this ligand movement results in an increase of the redox potential of heme 1, thus allowing the sequential uptake of the two electrons resulting from the conversion of the two thiosulfates to tetrathionate, leading to the reduction of both hemes; upon reduction, heme 2 undergoes a ligand switch, which increases its redox potential and hinders the back reaction. Most likely, HiPIP serves as the electron acceptor in vivo.