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

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.

The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity.

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.