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Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe–2S] cluster optical spectra and transfer chemistry

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Abstract

Human glutaredoxin 5 (Grx5) is one of the core components of the Isc (iron–sulfur cluster) assembly and trafficking machinery, and serves as an intermediary cluster carrier, putatively delivering cluster from the Isu scaffold protein to target proteins. The tripeptide glutathione is intimately involved in this role, providing cysteinyl coordination to the iron center of the Grx5-bound [2Fe–2S] cluster. Grx5 has a well-defined glutathione-binding pocket with protein amino acid residues providing many ionic and hydrogen binding contacts to the bound glutathione. In this report, we investigated the importance of these interactions in cluster chirality and exchange reactivity by systematically perturbing the crucial contacts by use of natural and non-natural amino acid substitutions to disrupt the binding contacts from both the protein and glutathione. Native Grx5 could be reconstituted with all of the glutathione analogs used, as well as other thiol ligands, such as DTT or l-cysteine, by in vitro chemical reconstitution, and the holo proteins were found to transfer [2Fe–2S] cluster to apo ferredoxin 1 at comparable rates. However, the circular dichroism spectra of these derivatives displayed prominent differences that reflect perturbations in local cluster chirality. These studies provided a detailed molecular understanding of glutathione–protein interactions in holo Grx5 that define both cluster spectroscopy and exchange chemistry.

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Acknowledgements

This work was supported by a Grant from the National Institutes of Health [AI072443].

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH, 43210, USA

    Sambuddha Sen & J. A. Cowan

  2. CIBIO, University of Trento, Via Sommarive 9, 38123, Povo, Italy

    Claudia Bonfio & Sheref S. Mansy

Authors
  1. Sambuddha Sen
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  2. Claudia Bonfio
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  3. Sheref S. Mansy
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  4. J. A. Cowan
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Contributions

SS and JAC designed the experiments and interpreted the results, CB designed and synthesized all the GSH analogs, SS performed the CD experiments. All the authors wrote the manuscript.

Corresponding author

Correspondence to J. A. Cowan.

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Sen, S., Bonfio, C., Mansy, S.S. et al. Investigation of glutathione-derived electrostatic and hydrogen-bonding interactions and their role in defining Grx5 [2Fe–2S] cluster optical spectra and transfer chemistry. J Biol Inorg Chem 23, 241–252 (2018). https://doi.org/10.1007/s00775-017-1525-5

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  • DOI: https://doi.org/10.1007/s00775-017-1525-5

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