Iron-sulfur (Fe-S) clusters are cofactors that are required for many biological processes. Conserved biosynthetic pathways synthesize Fe-S clusters and deliver clusters to target proteins using an elaborate distribution network. Both the cluster biosynthesis and transfer steps are tightly regulated to avoid the toxicity of labile iron and inorganic sulfide. The cysteine desulfurase, IscS, and the scaffold protein, IscU, constitute the core of the Fe-S assembly complex for the bacterial ISC pathway. IscS uses a pyridoxal 5'-phosphate cofactor to convert L-cysteine to L-alanine and mobilize sulfur as a persulfide intermediate for sulfur transfer to acceptor proteins. IscU combines this mobilized sulfur with ferrous iron and electrons to generate [2Fe-2S] clusters. [2Fe-2S]-IscU dissociates from IscS and participates in cluster transfer. Despite the importance of this pathway, the roles of additional protein components that interact with the assembly complex remain controversial, and mechanistic details for cluster synthesis and transfer are still poorly understood. Here, we take advantage of recently developed fluorescent reporter methodology to investigate the enzymology of the Fe-S cluster assembly reaction. A rhodamine fluorophore was used to label IscU and the decrease in fluorescence intensity (quenching) was used to monitor [2Fe-2S] cluster synthesis. The rate of [2Fe-2S]-IscU formation was determined as a function of each individual substrate (with the other substrates at high or saturating conditions) using a fluorescent plate reader located inside an anaerobic glovebox. Apo-IscU, an electron source (such as glutathione), ferrous iron, and L-cysteine were treated as substrates for the cluster assembly reaction. Some of the substrates demonstrated a hyperbolic relationship between the rate of the reaction and the concentration of substrate, and kinetic parameters were determined by fitting the data to a Michaelis-Menten equation. Other substrates did not exhibit saturation behavior. A model consistent with these kinetic results is described that provides insight into this complex reaction and a base line for future studies interrogating the role of additional components, such as CyaY, IscX, Fdx, and IscA, proposed to have a role in the synthesis or regulation of Fe-S cluster formation.
