Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis

Funding Information: We thank L?gia S. Nobre and Joana M. Baptista for contribution at the initial stage of the work, and Cl?udia S. Freitas for technical support. We also thank Professor Miguel Teixeira of ITQB-NOVA for critical reading of the manuscript. We thank the XALOC staff and floor coordinators at the synchrotron ALBA for the YtfEM data collection. We acknowledge the ESRF for provision of synchrotron radiation facilities and we would like to thank Gianluca Santoni for assistance using the beamline ID30A-3 for the YtfEM-E159L data collection. We also thank Diamond Light Source for beamtime and the staff of beamline I04 for assistance with crystal testing and data collection of YtfEM-E125L. Funding. This work was financially supported by Funda??o para a Ci?ncia e Tecnologia (Portugal) through fellowship SFRH/BD/118545/2016 (LOS) and R&D unit LISBOA-01-0145-FEDER007660 (MostMicro) co-funded by FCT/MCTES and FEDER funds under the PT2020 Partnership Agreement. This work was partially supported by PPBI ? Portuguese Platform of BioImaging (PPBI-POCI-01-0145-FEDER-022122) co-funded by national funds from OE ? ?Or?amento de Estado? and by European funds from FEDER ? ?Fundo Europeu de Desenvolvimento Regional.? We also acknowledge funding from the European Union?s Horizon 2020 Research and Innovation Program under grant agreement no. 810856. Publisher Copyright: © Copyright © 2021 Silva, Matias, Romão and Saraiva. Copyright: Copyright 2021 Elsevier B.V., All rights reserved.Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.publishersversionpublishe