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Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe–S Cluster Biosynthesis

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    Iron Insertion at the Assembly Site of the ISCU Scaffold Protein Is a Conserved Process Initiating Fe–S Cluster Biosynthesis
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    • Batoul Srour
      Batoul Srour
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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    • Sylvain Gervason
      Sylvain Gervason
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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    • Maren Hellen Hoock
      Maren Hellen Hoock
      Fachbereich Physik, Technische Universität Kaiserslautern, Erwin-Schrödinger-Str. 56, 67663 Kaiserslautern, Germany
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    • Beata Monfort
      Beata Monfort
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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    • Kristian Want
      Kristian Want
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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      Orcidhttps://orcid.org/0000-0001-7590-0896
    • Djabir Larkem
      Djabir Larkem
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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    • Nadine Trabelsi
      Nadine Trabelsi
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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    • Gautier Landrot
      Gautier Landrot
      Synchrotron SOLEIL, L’Orme des Merisiers, BP48 Saint Aubin 91192 Gif-Sur-Yvette, France
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      Orcidhttps://orcid.org/0000-0001-7673-421X
    • Andrea Zitolo
      Andrea Zitolo
      Synchrotron SOLEIL, L’Orme des Merisiers, BP48 Saint Aubin 91192 Gif-Sur-Yvette, France
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      Orcidhttps://orcid.org/0000-0002-2187-6699
    • Emiliano Fonda
      Emiliano Fonda
      Synchrotron SOLEIL, L’Orme des Merisiers, BP48 Saint Aubin 91192 Gif-Sur-Yvette, France
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    • Emilien Etienne
      Emilien Etienne
      Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), 31 Chemin Joseph Aiguier, 13402 Marseille, France
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      Orcidhttps://orcid.org/0000-0002-5999-7904
    • Guillaume Gerbaud
      Guillaume Gerbaud
      Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), 31 Chemin Joseph Aiguier, 13402 Marseille, France
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      Orcidhttps://orcid.org/0000-0002-7320-9839
    • Christina Sophia Müller
      Christina Sophia Müller
      Fachbereich Physik, Technische Universität Kaiserslautern, Erwin-Schrödinger-Str. 56, 67663 Kaiserslautern, Germany
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    • Jonathan Oltmanns
      Jonathan Oltmanns
      Fachbereich Physik, Technische Universität Kaiserslautern, Erwin-Schrödinger-Str. 56, 67663 Kaiserslautern, Germany
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    • Jesse B. Gordon
      Jesse B. Gordon
      Department of Chemistry, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218, United States
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      Orcidhttps://orcid.org/0000-0002-6331-7940
    • Vishal Yadav
      Vishal Yadav
      Department of Chemistry, The Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218, United States
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      Orcidhttps://orcid.org/0000-0002-7016-2991
    • Malgorzata Kleczewska
      Malgorzata Kleczewska
      Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland
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      Marcin Jelen
      Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Abrahama 58, 80-307 Gdansk, Poland
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    • Michel B. Toledano
      Michel B. Toledano
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
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      Rafal Dutkiewicz
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    • David P. Goldberg
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    • Volker Schünemann
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      Fachbereich Physik, Technische Universität Kaiserslautern, Erwin-Schrödinger-Str. 56, 67663 Kaiserslautern, Germany
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    • Bruno Guigliarelli
      Bruno Guigliarelli
      Aix Marseille Univ, CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines (BIP), 31 Chemin Joseph Aiguier, 13402 Marseille, France
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    • Bénédicte Burlat
      Bénédicte Burlat
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    • Christina Sizun
      Christina Sizun
      Institut de Chimie des Substances Naturelles, CNRS, Université Paris Saclay, Avenue de La Terrasse, 91190 Gif-sur-Yvette, France
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      Orcidhttps://orcid.org/0000-0002-5760-2614
    • Benoit D’Autréaux*
      Benoit D’Autréaux
      Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), 91198 Gif-sur-Yvette, France
      *Email: [email protected]
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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2022, 144, 38, 17496–17515
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.2c06338
    Published September 19, 2022
    Copyright © 2022 American Chemical Society
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    Abstract

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    Iron–sulfur (Fe–S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe–S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mössbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe–S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mössbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe–S cluster assembly process and establish that the initiation of Fe–S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c06338.

    • Calibration curves of SEC columns, Mössbauer, UV–visible, and CD spectra, structures of FeII model complexes, deconvoluted XANES spectra, and EPR temperature dependence studies; elution volumes and molecular weight determined by SEC, Mössbauer parameters, and absorption coefficients of the proteins at 280 nm; and determination of the ZFS parameters and analysis of the Mössbauer parameters (PDF)

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    Journal of the American Chemical Society

    Cite this: J. Am. Chem. Soc. 2022, 144, 38, 17496–17515
    Click to copy citationCitation copied!
    https://doi.org/10.1021/jacs.2c06338
    Published September 19, 2022
    Copyright © 2022 American Chemical Society
    Request reuse permissions

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