A persulfurated cysteine promotes active site reactivity in Azotobacter vinelandii rhodanese

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfu rtransferase (rhodanese) from Azotobacter vinelandii, have been investigate d through ligand binding, site-directed mutagenesis, and X-ray crystallogra phic techniques, in a combined approach. In native RhdA the active site Cys 230 is found persulfurated; fluorescence and sulfurtransferase activity mea surements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phospha te and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the cat alytic subunit of Cdc25 phosphatases share a common three-dimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants c arrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.