Gene/Protein Disease Symptom Drug Enzyme Compound
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Iron-sulfur (Fe-S) clusters are protein cofactors that are required for many essential cellular functions. Fe-S clusters are synthesized and inserted into target proteins by an elaborate biosynthetic process. The insensitivity of most Fe-S assembly and transfer assays requires high concentrations for components and places major limits on reaction complexity. Recently, fluorophore labels were shown to be effective at reporting cluster content for Fe-S proteins. Here, the incorporation of this labeling approach allowed the design and interrogation of complex Fe-S cluster biosynthetic reactions that mimic in vivo conditions. A bacterial Fe-S assembly complex, composed of the cysteine desulfurase IscS and scaffold protein IscU, was used to generate [2Fe-2S] clusters for transfer to mixtures of putative intermediate carrier and acceptor proteins. The focus of this study was to test whether the monothiol glutaredoxin, Grx4, functions as an obligate [2Fe-2S] carrier protein in the Fe-S cluster distribution network. Interestingly, [2Fe-2S] clusters generated by the IscS-IscU complex transferred to Grx4 at rates comparable to previous assays using uncomplexed IscU as a cluster source in chaperone-assisted transfer reactions. Further, we provide evidence that [2Fe-2S]-Grx4 delivers clusters to multiple classes of Fe-S targets via direct ligand exchange in a process that is both dynamic and reversible. Global fits of cluster transfer kinetics support a model in which Grx4 outcompetes terminal target proteins for IscU-bound [2Fe-2S] clusters and functions as an intermediate cluster carrier. Overall, these studies demonstrate the power of chemically conjugated fluorophore reporters for unraveling mechanistic details of biological metal cofactor assembly and distribution networks.
ACS Chem Biol 2016 11 18
PMID:Real-Time Kinetic Probes Support Monothiol Glutaredoxins As Intermediate Carriers in Fe-S Cluster Biosynthetic Pathways. 2765 19

Mitochondrial cysteine desulfurase is an essential component of the machinery for iron-sulfur cluster biosynthesis. It has been known that human cysteine desulfurase that is catalytically active in vitro can be prepared by overexpressing in Escherichia coli cells two protein components of this system, the cysteine desulfurase protein NFS1 and the auxiliary protein ISD11. We report here that this active preparation contains, in addition, the holo-form of E. coli acyl carrier protein (Acp). We have determined the stoichiometry of the complex to be [Acp]2:[ISD11]2:[NFS1]2. Acyl carrier protein recently has been found to be an essential component of the iron-sulfur protein biosynthesis machinery in mitochondria; thus, because of the activity of [Acp]2:[ISD11]2:[NFS1]2 in supporting iron-sulfur cluster assembly in vitro, it appears that E. coli Acp can substitute for its human homologue.
ACS Chem Biol 2017 04 21
PMID:Mitochondrial Cysteine Desulfurase and ISD11 Coexpressed in Escherichia coli Yield Complex Containing Acyl Carrier Protein. 2823 92

Iron-sulfur clusters are essential cofactors in many biochemical processes. ISD11, one of the subunits of the protein complex that carries out the cluster assembly in mitochondria, is necessary for cysteine desulfurase NFS1 stability and function. Several authors have recently provided evidence showing that ISD11 interacts with the acyl carrier protein (ACP). We carried out the coexpression of human mitochondrial ACP and ISD11 in E. coli. This work shows that ACP and ISD11 form a soluble, structured, and stable complex able to bind to the human NFS1 subunit modulating its activity. Results suggest that ACP plays a key-role in ISD11 folding and stability in vitro. These findings offer the opportunity to study the mechanism of interaction between ISD11 and NFS1.
ACS Chem Biol 2018 06 15
PMID:Rescuing the Rescuer: On the Protein Complex between the Human Mitochondrial Acyl Carrier Protein and ISD11. 2973 35