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Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction

Author

Listed:
  • Fabian Geissel

    (Comparative Biochemistry, RPTU Kaiserslautern)

  • Lukas Lang

    (Comparative Biochemistry, RPTU Kaiserslautern)

  • Britta Husemann

    (Comparative Biochemistry, RPTU Kaiserslautern)

  • Bruce Morgan

    (Saarland University)

  • Marcel Deponte

    (Comparative Biochemistry, RPTU Kaiserslautern)

Abstract

Glutaredoxins catalyze the reduction of disulfides and are key players in redox metabolism and regulation. While important insights were gained regarding the reduction of glutathione disulfide substrates, the mechanism of non-glutathione disulfide reduction remains highly debated. Here we determined the rate constants for the individual redox reactions between PfGrx, a model glutaredoxin from Plasmodium falciparum, and redox-sensitive green fluorescent protein 2 (roGFP2), a model substrate and versatile tool for intracellular redox measurements. We show that the PfGrx-catalyzed oxidation of roGFP2 occurs via a monothiol mechanism and is up to three orders of magnitude faster when roGFP2 and PfGrx are fused. The oxidation kinetics of roGFP2-PfGrx fusion constructs reflect at physiological GSSG concentrations the glutathionylation kinetics of the glutaredoxin moiety, thus allowing intracellular structure-function analysis. Reduction of the roGFP2 disulfide occurs via a monothiol mechanism and involves a ternary complex with GSH and PfGrx. Our study provides the mechanistic basis for understanding roGFP2 redox sensing and challenges previous mechanisms for protein disulfide reduction.

Suggested Citation

  • Fabian Geissel & Lukas Lang & Britta Husemann & Bruce Morgan & Marcel Deponte, 2024. "Deciphering the mechanism of glutaredoxin-catalyzed roGFP2 redox sensing reveals a ternary complex with glutathione for protein disulfide reduction," Nature Communications, Nature, vol. 15(1), pages 1-18, December.
    • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-45808-9
    DOI: 10.1038/s41467-024-45808-9
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    References listed on IDEAS

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    1. Patricia Begas & Linda Liedgens & Anna Moseler & Andreas J. Meyer & Marcel Deponte, 2017. "Glutaredoxin catalysis requires two distinct glutathione interaction sites," Nature Communications, Nature, vol. 8(1), pages 1-13, April.
    2. Linda Liedgens & Jannik Zimmermann & Lucas Wäschenbach & Fabian Geissel & Hugo Laporte & Holger Gohlke & Bruce Morgan & Marcel Deponte, 2020. "Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins," Nature Communications, Nature, vol. 11(1), pages 1-18, December.
    3. Daniel Trnka & Anna D. Engelke & Manuela Gellert & Anna Moseler & Md Faruq Hossain & Tobias T. Lindenberg & Luca Pedroletti & Benjamin Odermatt & João V. Souza & Agnieszka K. Bronowska & Tobias P. Dic, 2020. "Molecular basis for the distinct functions of redox-active and FeS-transfering glutaredoxins," Nature Communications, Nature, vol. 11(1), pages 1-12, December.
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