Assembly Mechanism Of [Fe₂S₂] Cluster In Ferredoxin From Acidithiobacillus Ferrooxidans

Ferredoxins are small, acidic, electron transfer proteins that are ubiquitous in biological redox systems. Among them, ferredoxins with one [Fe2S2] cluster per molecule are present in plants, animals, and bacteria, and form a distinct ferredoxin family, which is a typical [Fe2S2] cluster containing protein。Iron-sulfur clusters are one of the most ancient and ubiquitous redox centres in nature,They are involved in diverse physiological processes. However, the mechanism underlying the iron-sulfur-cluster assembly is still not fully understood so far. Recent studies have revealed that iron-sulflur cluster assembly in vivo mainly follows the "AUS" pathway, which requires scaffold proteins, and three important proteins (IscA, IscS and IscU) are involved in the case of iron-sulfur cluster assembly. Yet, the exact mechanism of iron-sulfur cluster assembly remains unclear.While various reports have already been published on the iron donor or scaffold protein involved in iron-sulfur cluster assembly, very little research has focused on the sulfur donor. L-allylglycine has been reported to be a suicide inhibitor of the cysteine desulfurase IscS/NifS by forming a covalent adduct with the active site cysteine of IscS/NifS, thereby inactivating the enzyme and blocking the AUS-dependent assembly pathway. Thus, selective inactivation of cysteine desulfurase can provide an efficient way to study the mechanism of iron-sulfur cluster assembly in vivo.This study investigates the in vitro assembly of a [Fe2S2] cluster in the ferredoxin from Acidithiobacillus ferrooxidans in the presence of three scaffold proteins:IscA, IscS, and IscU. The Nondenaturing PAGE, UV-vis scanning, EPR spectra and MALDI-TOF MS results of the reconstitute ferredoxin confirmed that the iron-sulfur cluster was correctly assembled into the protein. Inactivation of cysteine desulfurase by L-allylglycine completely blocked the [Fe2S2] cluster assembly in ferredoxin in E. coli. Analysis of the IscS activity by L-allylglycine in vitro and UV-vis scanning of ferredoxin produced with or without addition of L-allylglycine to the culture, confirmed that the cysteine desulfurase is indeed an essential component for the iron-sulfur cluster assembly. The results provide strong evidences that the assembly of the [Fe2S2] cluster in ferredoxin follows the AUS pathway.