Quantitative assessment of the determinant structural differences between redox-active and inactive glutaredoxins

Linda Liedgens,Bruce Morgan,Fabian Geissel,Hugo Laporte,Jannik Zimmermann,Marcel Deponte,Lucas Wäschenbach,Holger Gohlke

doi:10.1038/s41467-020-15441-3

Abstract

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Enzymatically active glutaredoxins harbor both a glutathione-scaffold site for reacting with glutathionylated disulfide substrates and a glutathione-activator site for reacting with reduced glutathione. Here, using yeast ScGrx7 as a model protein, we comprehensively identified and characterized key residues from four distinct protein regions, as well as the covalently bound glutathione moiety, and quantified their contribution to both interaction sites. Additionally, we developed a redox-sensitive GFP2-based assay, which allowed the real-time assessment of glutaredoxin structure-function relationships inside living cells. Finally, we employed this assay to rapidly screen multiple glutaredoxin mutants, ultimately enabling us to convert enzymatically active and inactive glutaredoxins into each other. In summary, we have gained a comprehensive understanding of the mechanistic underpinnings of glutaredoxin catalysis and have elucidated the determinant structural differences between the two main classes of glutaredoxins.

Highlights

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins
We recently confirmed that enzymatically active Grx have two distinct glutathione-interaction sites, one glutathione-scaffold site that interacts with glutathionylated disulfide substrates (GSSR)
Whilst yeast do not harbor any bona fide glutathione peroxidase, it was recently shown that GSH and/or Grx can reduce both 1-Cys and typical 2-Cys peroxiredoxins thereby leading to GSSG production[42,46,47,49,50]

Summary

Introduction

Class I glutaredoxins are enzymatically active, glutathione-dependent oxidoreductases, whilst class II glutaredoxins are typically enzymatically inactive, Fe-S cluster-binding proteins. Active Grx use glutathione as a substrate[3,4,7,8,9,10] (Fig. 1a, b), whereas inactive Grx use glutathione as a ligand for their iron–sulfur cluster[1,2,5,6] (Fig. 1c) How both Grx subfamilies exert their nonredundant physiological functions in redox catalysis and iron metabolism remains puzzling, and the underlying structure–function relationships are only partially understood. During the oxidative half-reaction, and one glutathione-activator site that interacts with reduced glutathione (GSH) during the reductive half-reaction[7,11,12] (Fig. 1a) Based on these results, we hypothesized that modified glutathione interactions underlie the enzymatic inactivity of class II Grx and kinetically uncouple these proteins from the glutathione pool[7,12,13]. The aim of this work was, first, to identify protein areas that are relevant for redox catalysis and that are modified in enzymatically inactive Grx and, second, to quantitatively assess the contribution of these protein areas toward the oxidoreductase a GSH

Methods

Results

Conclusion