CyaY and TusA regulate ISC- and SUF-mediated l-cysteine desulfurase activity.

CyaY, the frataxin homolog of , plays an important role in ISC iron-sulfur cluster assembly through interactions with the cysteine desulfurase IscS, which regulate the supply of sulfur. IscS is not exclusive for ISC Fe-S cluster assembly, as it functions as a hub for the supply of sulfur to a number of other sulfur-requiring pathways, such as for the biosynthesis of Moco and thiolated tRNAs. How the balance of sulfur supply to the various competing pathways is achieved is not fully understood, but a network of protein-protein interactions plays a key role. For example, IscU and TusA compete for binding to IscS and thus for sulfur supply to ISC and Moco/tRNA biosynthesis. Here, we show that TusA can displace CyaY from IscS and can form hetero-complexes involving IscS, CyaY and TusA. Displacement of CyaY from IscS raised the question of whether it can interact with the SUF pathway. The SUF cysteine desulfurase SufS functions as a complex with SufE. Native mass spectrometry studies showed that the SufS dimer can bind up to four SufE molecules, two at high affinity, and two at low affinity, sites. Titration of SufSE (or SufS alone) with CyaY demonstrated binding, probably at the lower affinity site in competition with SufE. Binding of CyaY dramatically reduced the activity of SufSE , and over-expression of CyaY also significantly affected total cellular desulfurase activity and Fe-S cluster assembly, with the greatest effect observed in mutant strains in which SufS was the principal desulfurase. These data point to a physiological role for CyaY in regulating the desulfurase activity of IscS and SufS and, hence, both the iron-sulfur assembly systems. They also demonstrate that TusA can displace the regulatory CyaY protein from IscS-CyaY complexes, facilitating sulfur delivery from IscS to other essential cellular processes, and increasing the likelihood of SufSE-CyaY interactions.