Azotobacter vinelandii rhodanese: selenium loading and ion interaction studies

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Rhodanese is a sulfurtransferase catalyzing sulfur transfer from thiosulfate to cyanide.
• Prokaryotic rhodanese-like proteins from Azotobacter vinelandii possess similar enzymatic activity.

Purpose of the Study:

• To investigate the ionic interactions and reaction mechanisms of Azotobacter vinelandii rhodanese-like protein.
• To elucidate the role of the catalytic cysteine residue in sulfur and selenium transfer.
• To explore the potential in vivo function of rhodanese-like proteins in selenium delivery.

Main Methods:

• Selective labeling of the catalytic Cys230 residue with [15N]Cys.
• Monitoring changes in 1H and 15N NMR resonances upon ion addition.
• Utilizing fluorescence spectroscopy to observe selenium loading.
• Analyzing the 1H-NMR spectrum for enzyme status and sulfur loading.

Main Results:

• Sulfur transfer is mediated by a specific reaction of the persulfurated cysteine residue with sulfur acceptors, independent of anion presence.
• A specific region in the 1H-NMR spectrum directly indicates enzyme status and sulfur loading.
• Selenium loading by selenodiglutathione involves direct interaction with the catalytic cysteine residue.
• Rhodanese-like proteins are implicated in the in vivo delivery of reactive selenium.

Conclusions:

• The catalytic mechanism of sulfur transfer by rhodanese-like proteins involves direct interaction of the persulfurated cysteine residue with acceptors.
• NMR and fluorescence spectroscopy are effective tools for monitoring enzyme activity and substrate loading.
• Rhodanese-like proteins play a crucial role in selenium metabolism and delivery within biological systems.