Two novel monothiol glutaredoxins from yeast (ScGrx6 and ScGrx7) were identified and analyzed in vitro. Both proteins are highly suited to study structure-function relationships of glutaredoxin subclasses because they differ from all monothiol glutaredoxins investigated so far and share features with dithiol glutaredoxins. ScGrx6 and ScGrx7 are, for example, the first monothiol glutaredoxins showing an activity in the standard glutaredoxin transhydrogenase assay with glutathione and bis-(2-hydroxyethyl)-disulfide. Steady-state kinetics of ScGrx7 with glutathione and cysteine-glutathione disulfide are similar to dithiol glutaredoxins and are consistent with a ping-pong mechanism. In contrast to most other glutaredoxins, ScGrx7 and ScGrx6 are able to dimerize noncovalently. Furthermore, ScGrx6 is the first monothiol glutaredoxin shown to directly bind an iron-sulfur cluster. The cluster can be stabilized by reduced glutathione, and its loss results in the conversion of tetramers to dimers. ScGrx7 does not bind metal ions but can be covalently modified in Escherichia coli leading to a mass shift of 1090 +/- 14 Da. What might be the structural requirements that cause the different properties? We hypothesize that a G(S/T)x3 insertion between a highly conserved lysine residue and the active site cysteine residue could be responsible for the abrogated transhydrogenase activity of many monothiol glutaredoxins. In addition, we suggest an active site motif without proline residues that could lead to the identification of further metal binding glutaredoxins. Such different properties presumably reflect diverse functions in vivo and might therefore explain why there are at least seven glutaredoxins in yeast.

Two dithiol glutaredoxins (Grxs), Grx1 and Grx2, from yeast have been characterized to date. A third putative dithiol glutaredoxin-encoding gene (GRX8) has been identified in silico. Here we show that deletion of GRX8 does not result in a reduced growth rate under oxidative stress conditions, nor does it enhance the defects of Deltagrx1 and Deltagrx2 single or double mutants. We furthermore compare the enzymatic properties of recombinant ScGrx8 with the monothiol glutaredoxin ScGrx7. Molecular models of ScGrx8 suggest that the protein has a canonical Grx fold, a significantly altered substrate binding site, and a Trp14-type cysteine motif at the catalytic center. ScGrx8 did not bind heavy metal ions and was exclusively monomeric. Apparent k(cat) values for ScGrx8 in the standard enzymatic assay were about 3 orders of magnitude less than for ScGrx7, whereas apparent K(m) values were comparable. Mass spectrometric analyses support a ping-pong mechanism for ScGrx7 and ScGrx8 with a glutathionylated protein as an intermediate. Reduction kinetics of ScGrx8 disulfide, glutathionylated ScGrx8(C28S), and glutathionylated ScGrx7 revealed significant differences between the proteins. Surprisingly, mutation of the more C-terminal cysteine residue in the CPDC motif of ScGrx8 also abolished the slight enzymatic activity, and thus the standard catalytic mechanism for glutathionylated substrates does not apply to the enzyme. In summary, ScGrx8 has several novel structural and mechanistic features expanding the subclasses of glutaredoxins. A refined catalytic model for monothiol and dithiol glutaredoxins is presented explaining the diversity of enzymatic activities in vitro and pointing to different functions in vivo.

Mitochondrial protein import (MPI) is essential for the biogenesis of mitochondria in all eukaryotes. Current models of MPI are predominantly based on experiments with one group of eukaryotes, the opisthokonts. Although fascinating genome database-driven hypotheses on the evolution of the MPI machineries have been published, previous experimental research on non-opisthokonts usually focused on the analysis of single pathways or components in, for example, plants and parasites. In this study, we have established the kinetoplastid parasite Leishmania tarentolae as a model organism for the comprehensive analysis of non-opisthokont MPI into all four mitochondrial compartments. We found that opisthokont marker proteins are efficiently imported into isolated L. tarentolae mitochondria. Vice versa, L. tarentolae marker proteins of all compartments are also imported into mitochondria from yeast. The results are remarkable because only a few of the more than 25 classical components of the opisthokont MPI machineries are found in parasite genome databases. Our results demonstrate that different MPI pathways are functionally conserved among eukaryotes despite significant compositional differences of the MPI machineries. Moreover, our model system could lead to the identification of significantly altered or even novel MPI components in non-opisthokonts. Such differences might serve as starting points for drug development against parasitic protists.