Dissecting the molecular interactions of mitochondrial glutaredoxin S15 in plants – mitoglu

The biochemical, spectroscopic and structural analysis of GRXS15 holoforms obtained by in vitro anaerobic reconstitution will allow determining the oligomerisation status and nature of the assembled ISCs. On the basis of structural alignments and using targeted mutagenesis and combinatorial approaches, we will generate new knowledge on the structure-function relationship of this GRX and of proteins of the same class. The properties of these variants will be determined (i) by analysing their ability to bind an ISC and its lability, (ii) by finely examining protein-protein interactions with known or newly identified partner proteins, (iii) by performing heterologous expression in a yeast mutant deficient in mitochondrial Grx5 and (iv) by assessing their possible redox properties using in vitro activity assays with roGFP2 and oxidation sensitivity tests.

We have expressed both tagged and untagged recombinant proteins in E. coli in full-length or in a shorter form missing an N-terminal part that is supposed to be disordered, as well as some mutated variants. It took quite some time to obtain soluble, non-aggregated protein forms with sufficient yield. For that we have tested many expression strains, culture conditions and purification procedures. We were able to determine that the pKa for the sole cysteine 91 of AtGRXS15 was of 5.5. This indicates that it is likely reactive and susceptible to oxidation. Hence, we observed that H2O2 promoted the formation of dimers and the effect is concentration dependent and that GSSG and GSNO induced glutathionylation of the protein. The ability of mitochondrial thioredoxins to reduce oxidized GRXS15 was determined in vitro. By purifying apo-forms, we have been able to set up an in vitro procedure for the reconstitution of the iron-sulfur cluster in GRXS15 under anaerobiosis. This will allow us to start the structural analyses.
Concerning the physiological analyses, it is worth noting that Arabidopsis grxs15 null mutants are not viable, but mutants complemented with the variant GRXS15 K83A develop with a dwarf phenotype similar to knockdown line GRXS15amiR. In an in-depth metabolic analysis of the variant and knockdown GRXS15 lines, we showed that most Fe-S cluster-dependent processes are not affected, including biotin biosynthesis, molybdenum cofactor biosynthesis, the electron transport chain and aconitase in the TCA cycle. Instead, we observed an increase in most TCA cycle intermediates and amino acids, especially pyruvate, glycine and branched-chain amino acids (BCAAs). Additionally, we found an accumulation of branched-chain a-keto acids (BCKAs), the first degradation products resulting from transamination of BCAAs, all pointing to lypoylation synthesis defect.

The next steps are:
1) To better understand the structural features of Arabidopsis GRXS15 that condition the degree of complementation of the yeast ?grx5 mutant and its oxidoreductase activity
2) To further analyse the impact of GRXS15 deficiency on mitochondrial and cytosolic Fe-S-dependent enzymes and physiological consequences at the whole plant level
3) To dissect the role of GRXS15 in the GSH-dependent transfer of ISC to acceptor proteins, that have to be identified.

1. Przybyla-Toscano J, Christ L, Keech O, Rouhier N. Iron-sulfur proteins in plant mitochondria: roles and maturation. J Exp Bot. 2021, 72(6):2014-2044. doi: 10.1093/jxb/eraa578.
2. Berndt C, Christ L, Rouhier N, Mühlenhoff U. Glutaredoxins with iron-sulphur clusters in eukaryotes - Structure, function and impact on disease. Biochim Biophys Acta Bioenerg. 2021, 1862(1):148317.
3. Moseler A, Kruse I, Maclean AE, Pedroletti L, Franceschetti M, Wagner S, Wehler R, Fischer-Schraderg K, Poscheth G, Wirtz M, Dörmann P, Hildebrandt TM, Hell R, Schwarzländer M, Balk J, Meyer AJ. The function of glutaredoxin GRXS15 is required for lipoyl-dependent dehydrogenases in mitochondria. Plant physiology in press

Several metabolic pathways and cellular processes in plants depend on the functioning of iron-sulfur (Fe-S) proteins, whose cofactors are assembled through dedicated assembly machineries present in the cytosol, plastids and mitochondria. To cite only a few examples, Fe- S proteins are present in the photosynthetic and respiratory electron transfer chains and they are needed for sulfur and nitrogen assimilation, or co-enzyme synthesis such as biotin and lipoic acid. In plants as in other organisms, the incorporation of Fe-S clusters into proteins
requires first the de novo assembly of iron-sulfur clusters (ISCs) onto scaffold proteins and their transfer to acceptor proteins via the action of several maturation factors, and among those class II glutaredoxins (GRXs). The recent demonstration that GRXS15 coordinates an [2Fe-2S] cluster using glutathione molecules which can be transferred to an acceptor protein and that null mutants for the mitochondrial GRXS15 in Arabidopsis are embryo-lethal provided clear evidence that GRXS15 is an essential component of the ISC transfer machinery. This finding opens a new avenue towards molecular understanding of how mitochondrial Fe-S proteins are assembled. The proposed project aims primarily at dissecting the role of GRXS15 in the transfer of ISCs to target proteins in Arabidopsis. The biochemical, spectroscopic and structural analysis of GRXS15 holoforms obtained by in vitro anaerobic reconstitution will allow determining the oligomerisation status and nature of the assembled ISCs. On the basis of structural alignments and using targeted mutagenesis and combinatorial approaches, we will generate new knowledge on the structure-function relationship of this GRX and of proteins of the same class. The properties of these variants will be determined (i) by analysing their ability to bind an ISC and its lability, (ii) by finely examining protein-protein interactions with known or newly identified partner proteins, (iii) by performing heterologous expression in a yeast mutant deficient in mitochondrial Grx5 and (iv) by assessing their possible redox properties using in vitro activity assays with roGFP2 and oxidation sensitivity tests. Following earlier work, grxs15 null mutants partially rescued through expression of heterologous GRXs or mutated GRXS15 are expected to display distinct developmental and physiological phenotypes. Thus, it will be investigated whether these phenotypes are related to specific Fe-S enzyme defects and whether a bottleneck in mitochondrial ISC transfer is restricted to mitochondrial target proteins or whether it also affects cytosolic metalloenzymes. This could be because a yet unknown mitochondrial sulfur-containing compound is known to be exported for the maturation of cytosolic and nuclear proteins, and because synthesis of the molybdenum cofactor found in several cytosolic Fe-S enzymes requires a mitochondrial Fe-S enzyme.