Surface changes and role of buried water molecules during the sulfane sulfur transfer in rhodanese from Azotobacter vinelandii : a fluorescence quenching and nuclear magnetic relaxation dispersion spectroscopic study

The Azotobacter vinelandii rhodanese is a sulfurtransferase Enz. that catalyzes the Transf. of the outer sulfur atom from thiosulfate to cyanide. Recently, investigations by NMR relaxation on the 15N-enriched protein reported that interdomain contacts are rigidly maintained upon the sulfane sulfur Transf. from the Enz. to the substrate. The modality of the enzymatic mechanism is then confined to a Surf. interaction, including Dynam. of water molecules buried in the Tert. structure. Thus, investigations have been carried out by fluorescence, circular dichroism,/Nucl. magnetic relaxation dispersion measurements. The comparison of circular dichroism spectra of the persulfurated Enz. /the sulfur-free form indicated that small changes occur. Fluorescence quenching Studs. have been performed to evaluate the conformational changes during Catalys. using the fluorescent probe 8-anilinonaphthalene-2-sulfonic acid, and acrylamide, iodide, and cesium ions as quenchers. Changes in exchange dynamics of water molecules buried in the structure with bulk water, observed by nuclear magnetic relaxation dispersion, are due to local conformational transitions, likely involving residues around the active site, and are consistent with the global correlation time found by 15N relaxation. These results, taken together, provide important information for elucidating the conformational features of the mechanism of action of the enzyme either in the role of a selective donor of a sulfur atom to small-sized substrates (i.e., to cyanide, transforming it into thiocyanate) or in the role of sulfur insertase for the formation of the Fe2S2 iron-sulfur cluster in sulfur-deprived ferredoxins.