Abstract
Bifunctional dihaem cytochrome c thiosulfate dehydrogenases/tetrathionate reductases (TsdA) exhibit different catalytic properties depending on the source organism. In the human food-borne intestinal pathogen Campylobacter jejuni, TsdA functions as a tetrathionate reductase enabling respiration with tetrathionate as an alternative electron acceptor. In the present study, evidence is provided that Cys138 and Met255 serve as the sixth ligands of Haem 1 and Haem 2 respectively, in the oxidized CjTsdA wt protein. Replacement of Cys138 resulted in a virtually inactive enzyme, confirming Haem 1 as the active site haem. Significantly, TsdA variants carrying amino acid exchanges in the vicinity of the electron-transferring Haem 2 (Met255, Asn254 and Lys252) exhibited markedly altered catalytic properties of the enzyme, showing these residues play a key role in the physiological function of TsdA. The growth phenotypes and tetrathionate reductase activities of a series of ΔtsdA/*tsdA complementation strains constructed in the original host C. jejuni 81116, showed that in vivo, the TsdA variants exhibited the same catalytic properties as the pure, recombinantly produced enzymes. However, variants that catalysed tetrathionate reduction more effectively than the wild-type enzyme did not allow better growth.