Mechanistic Insights of IscU Conformation Regulation for Fe–S Cluster Biogenesis Revealed by Variable-Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry

Iron-sulfur (Fe-S) clusters are necessary for essential cellular processes such as oxidative respiration. In the householding ISC Fe-S biosynthetic pathway, the cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, in which Fe–S clusters are assembled. Previous structural studies by crystallography and nuclear magnetic resonance (NMR) spectroscopy suggest that metamorphic IscU exists in at least a structured form and a disordered form, and switch conformation upon metal or IscS binding. Yet, there is debate on which form binds IscS and participates in Fe-S assembly. Also, previous NMR and circular dichroism spectroscopy results reveal the cold-denaturation of IscU and the effects of metal binding on cold-denaturation. The recent development of variable-temperature electrospray ionization (vT-ESI) native ion mobility mass spectrometry (nIM-MS) enables unprecedented informative studies that contain binding, structure, and temperature dependence in a single experiment. Here, vT-ESI nIM-MS is implemented to thoroughly investigate structure regulations of IscU and relations to Fe-S assembly. The results show clear cold/heat-denaturation of IscU as IscU shifts towards high charge states that have more extended conformation under extreme temperatures. Comparison of Zn-IscU and apo-IscU reveals that (i) Zn(II) binding attenuates the cold/heat-denaturation of IscU and promotes the refolding of IscU; (ii) Zn(II) binding prevents IscU shifting towards disordered high charge states, and only structured and intermediate forms bind Zn(II); and (iii) Zn(II) binding attenuates collisional induced unfolding (CIU) of the intermediate form. In the solution containing IscS, the temperature-dependent and chelator-dependent Zn(II) loss of IscU facilitates CIU of the intermediate form of IscU. Overall, these results suggest Zn(II) stabilizes and restricts the structural flexibility of IscU towards structure and intermediate forms. IscS binding loosens the intermediate form IscU active site local conformation to release Zn(II) for cluster assembly.