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 realtime 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.

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So-called 1-Cys peroxiredoxins (Prx) employ only one cysteine residue for the reduction of hydroperoxides and require an external thiol for the reduction of a reactive sulfenic acid during the catalytic cycle. Hence, 1-Cys Prx, which often belong to the structural Prx5-or the Prx6-type subfamily, are potentially promiscuous enzymes that could react with a variety of thiols. Furthermore, the dependence on an external thiol could affect the susceptibility of 1-Cys Prx to hyperoxidation, i.e., the formation of a sulfinic or sulfonic acid. Here, we compared the reaction mechanisms and kinetics of the Prx5-and Prx6-type enzymes PfAOP and PfPrx6 from the malaria parasite Plasmodium falciparum to address the hyperoxidation susceptibility and potential substrate promiscuity of 1-Cys Prx. While PfAOP did not react with common thiol-disulfide oxidoreductases, the enzyme turned out to be promiscuous regarding the reduction by synthesized glutathione analogues and other low-molecular-weight thiols. Furthermore, we established a complete single turnover experiment for PfAOP with glutathione and the glutaredoxin PfGrx and identified the rapid H 2 O 2 -dependent hyperoxidation of PfAOP as the cause for the apparent preference of this Prx5-type enzyme for alkylhydroperoxides in vitro. Unlike promiscuous PfAOP, PfPrx6 was inactive with ascorbate, the physiological low-molecular-weight thiols glutathione, cysteine, cysteamine, coenzyme A, and dihydrolipoamide, as well as physiological protein thiols, including PfTrx1, PfGrx, and the resolving cysteine of the Prx1-type enzyme PfPrx1a in potential hetero-oligomers. Reduction of PfPrx6 was only observed with dithiothreitol and required the presence of a histidine residue, which protects the enzyme from hyperoxidation and is the major structural difference between the active sites of Prx5-and Prx6-type enzymes. We propose two alternative evolutionary adaptations of the 1-Cys Prx mechanism to hyperoxidation and the formation of alternative mixed disulfides that could explain the co-existence of promiscuous Prx5-and protected Prx6-type enzymes in a variety of organisms and subcellular compartments.

Mitochondria play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here, we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenic enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and increases the levels of its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in compromised protein import into mitochondria and reduced steady-state levels of mitochondrial proteins. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery to improve mitochondrial protein import during phases of metabolic transition.

Type II NADH dehydrogenases (NDH2) are monotopic enzymes present in the external or internal face of the mitochondrial inner membrane that contribute to NADH/NAD+ balance by conveying electrons from NADH to ubiquinone without coupled proton translocation. Herein, we characterize the product of a gene present in all species of the human protozoan parasite Leishmania as a bona fide, matrix-oriented, type II NADH dehydrogenase. Within mitochondria, this respiratory activity concurs with that of type I NADH dehydrogenase (complex I) in some Leishmania species but not others. To query the significance of NDH2 in parasite physiology, we attempted its genetic disruption in two parasite species, exhibiting a silent (Leishmania infantum, Li) and a fully operational (Leishmania major, Lm) complex I. Strikingly, this analysis revealed that NDH2 abrogation is not tolerated by Leishmania, not even by complex I–expressing Lm species. Conversely, complex I is dispensable in both species, provided that NDH2 is sufficiently expressed. That a type II dehydrogenase is essential even in the presence of an active complex I places Leishmania NADH metabolism into an entirely unique perspective and suggests unexplored functions for NDH2 that span beyond its complex I–overlapping activities. Notably, by showing that the essential character of NDH2 extends to the disease-causing stage of Leishmania, we genetically validate NDH2—an enzyme without a counterpart in mammals—as a candidate target for leishmanicidal drugs.

Mitochondria are essential organelles that play a key role in cellular energy metabolism. Transitions between glycolytic and respiratory conditions induce considerable adaptations of the cellular proteome. These metabolism-dependent changes are particularly pronounced for the protein composition of mitochondria. Here we show that the yeast cytosolic ubiquitin conjugase Ubc8 plays a crucial role in the remodeling process when cells transition from respiratory to fermentative conditions. Ubc8 is a conserved and well-studied component of the catabolite control system that is known to regulate the stability of gluconeogenesis enzymes. Unexpectedly, we found that Ubc8 also promotes the assembly of the translocase of the outer membrane of mitochondria (TOM) and stabilizes its cytosol-exposed receptor subunit Tom22. Ubc8 deficiency results in a compromised protein import into mitochondria and a subsequent accumulation of mitochondrial precursor proteins in the cytosol. Our observations show that Ubc8, which is controlled by the prevailing metabolic conditions, promotes the switch from glucose synthesis to glucose usage in the cytosol and induces the biogenesis of the mitochondrial TOM machinery in order to improve mitochondrial protein import during phases of metabolic transition.