Abstract
In plants, the mitochondrial thioredoxin (TRX) system generally comprises only one or two isoforms belonging to the TRX h or o classes, being less well developed compared to the numerous isoforms found in chloroplasts. Unlike most other plant species, Arabidopsis thaliana possesses two TRXo isoforms whose physiological functions remain unclear. Here, we performed a structure–function analysis to unravel the respective properties of the duplicated TRXo1 and TRXo2 isoforms. Surprisingly, when expressed in Escherichia coli, both recombinant proteins existed in an apo-monomeric form and in a homodimeric iron–sulfur (Fe-S) cluster-bridged form. In TRXo2, the [4Fe-4S] cluster is likely ligated in by the usual catalytic cysteines present in the conserved Trp-Cys-Gly-Pro-Cys signature. Solving the three-dimensional structure of both TRXo apo-forms pointed to marked differences in the surface charge distribution, notably in some area usually participating to protein–protein interactions with partners. However, we could not detect a difference in their capacity to reduce nitrogen-fixation-subunit-U (NFU)-like proteins, NFU4 or NFU5, two proteins participating in the maturation of certain mitochondrial Fe-S proteins and previously isolated as putative TRXo1 partners. Altogether, these results suggest that a novel regulation mechanism may prevail for mitochondrial TRXs o, possibly existing as a redox-inactive Fe-S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation in specific physiological conditions.
Highlights
Mitochondria are important organelles being notably the site of production of cellular energy in the form of adenosine triphosphate (ATP) through the process of oxidative phosphorylation and being an important site for the amino acid and lipid metabolisms and for the biosynthesis of many crucial vitamins and cofactors
Arabidopsis thaliana TRXo Isoforms Exist in Two Forms upon Expression in E. coli
To characterize the structure–function relationship of TRXo1 and TRXo2, the mature forms of these proteins were expressed in E. coli as untagged and His-tagged recombinant proteins by removing respectively 82 and 47 residues at the N-terminus constituting their putative mitochondrial targeting sequence
Summary
Mitochondria are important organelles being notably the site of production of cellular energy in the form of adenosine triphosphate (ATP) through the process of oxidative phosphorylation and being an important site for the amino acid and lipid metabolisms and for the biosynthesis of many crucial vitamins and cofactors. Antioxidants 2018, 7, 142; doi:10.3390/antiox7100142 www.mdpi.com/journal/antioxidants (GPXL) families [4] These thiol peroxidases use reactive cysteine residues for catalysis and rely either on a glutathione/glutaredoxin (GSH/GRX) system or on a thioredoxin (TRX) system for their regeneration [5,6,7,8]. At the level of some sensitive protein cysteine residues, this leads to reversible modifications such as the formation of sulfenic acid, of disulfide bonds, or of glutathionylated or nitrosylated cysteines In this respect, it is surprising that the systems devoted to the reduction of these oxidized cysteine forms are not very developed in mitochondria. Unlike most other plant species, A. thaliana possesses two TRXo isoforms which add to TRXh2 [15] Their regeneration should be in principle dependent on the nicotinamide adenine dinucleotide phosphate reduced (NADPH)-thioredoxin reductase (NTR) A and/or B [13,16]
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