tailieunhanh - Báo cáo khoa học: Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies

Rhodanese is a sulfurtransferase whichin vitrocatalyzes the transfer of a sulfane sulfur fromthiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandiiwere studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using [ 15 N]Cys, and changes in 1 Hand15 NNMRresonances onadditionof different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions | Eur. J. Biochem. 270 4208-4215 2003 FEBS 2003 doi Azotobacter vinelandii rhodanese Selenium loading and ion interaction studies Sonia Melino1 Daniel O. Cicero1 2. Maria Orsale1 Fabio Forlani3 Silvia Paaani3 and Maurizio Paci1 2 1 Dipartimento di Scienze e Tecnologie Chimiche and 2INFM Sez. B University of Rome Tor Vergata Italy 3Dipartimento di Scienze Molecolari Agroalimentari University of Milan Italy Rhodanese is a sulfurtransferase which in vitro catalyzes the transfer of a sulfane sulfur from thiosulfate to cyanide. Ionic interactions of the prokaryotic rhodanese-like protein from Azotobacter vinelandii were studied by fluorescence and NMR spectroscopy. The catalytic Cys230 residue of the enzyme was selectively labelled using 15N Cys and changes in 1Hand 15N NMR resonances on addition of different ions were monitored. The results clearly indicate that the sulfur transfer is due to a specific reaction of the persulfurated Cys residue with a sulfur acceptor such as cyanide and not to the presence of the anions. Moreover the 1H-NMR spectrum of a defined spectral region is indicative of the status of the enzyme and can be used to directly monitor sulfur loading even at low concentrations. Selenium loading by the addition of selenodiglutathione was monitored by fluorescence and NMR spectroscopy. It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme. These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo. Keywords 15N-NMR Azotobacter vinelandii rhodanese selenodiglutathione sulfurtransferase. The rhodanese from Azotobacter vinelandii RhdA is a sulfurtransferase which catalyzes in vitro the production of thiocyanate transferring the sulfane sulfur atom from thiosulfate to cyanide by a double displacement mechanism thiosulfate-cyanide sulfurtransferase EC 1-3 . The best studied rhodanese is that from