Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic...

Area of Science:

• Biochemistry
• Molecular Biology
• Bioinorganic Chemistry

Background:

• Iron-sulfur (Fe-S) clusters are vital protein cofactors essential for numerous cellular processes.
• Fe-S cluster biosynthesis and insertion into proteins involve complex pathways.
• Existing assays for Fe-S assembly and transfer are limited by high component concentrations and reaction complexity.

Purpose of the Study:

• To investigate the role of the monothiol glutaredoxin, Grx4, as an intermediate carrier in the Fe-S cluster distribution network.
• To design and analyze complex Fe-S cluster biosynthetic reactions under conditions mimicking in vivo environments.
• To leverage fluorophore labeling for enhanced detection and mechanistic studies of Fe-S cluster transfer.

Main Methods:

• Utilized a bacterial Fe-S assembly complex (IscS-IscU) to generate [2Fe-2S] clusters.
• Employed fluorophore labeling to monitor Fe-S cluster content in proteins.
• Assessed the transfer of [2Fe-2S] clusters from the IscS-IscU complex to Grx4 and other Fe-S target proteins.
• Analyzed cluster transfer kinetics using global fitting to model the distribution network.

Main Results:

• [2Fe-2S] clusters generated by the IscS-IscU complex transferred efficiently to Grx4.
• Evidence suggests [2Fe-2S]-Grx4 delivers clusters to multiple targets via dynamic and reversible ligand exchange.
• Kinetic analysis supports a model where Grx4 outcompetes terminal targets for IscU-bound [2Fe-2S] clusters.
• Fluorophore reporters proved effective for studying complex Fe-S cluster assembly and distribution.

Conclusions:

• Grx4 functions as an essential intermediate carrier protein in the Fe-S cluster distribution pathway.
• The study highlights the dynamic and reversible nature of Fe-S cluster transfer mediated by Grx4.
• Fluorophore labeling is a powerful tool for elucidating the mechanisms of biological metal cofactor metabolism.