The role of glutaredoxin S15 in the transfer of Fe–S clusters and their consumption by lipoyl synthase in Arabidopsis mitochondria

Abstract

Iron–sulfur (Fe–S) clusters are vital cofactors in all domains of life. The mitochondrial Fe–S cluster biogenesis occurs through two major steps: the initial construction of [2Fe–2S] clusters, followed by their assembly into [4Fe–4S] clusters. However, how the clusters are distributed among the respective apoproteins is still not fully understood. In the presented thesis, we investigate the Fe–S cluster-dependent metabolism within plant mitochondria, using <em>Arabidopsis thaliana</em> as a model organism. <br /> First, we summarise the existing knowledge on Fe–S cluster biogenesis and trafficking. We then discuss the metabolic consequences of compromised Fe–S cluster availability. In addition, we speculate about the potential release of sulfide and estimate the amount that might be set free by the continuous turnover of Fe–S cluster-containing proteins. <br /> Furthermore, we studied the function of the glutaredoxin S15 (GRXS15). In contrast with other organisms, plants contain only GRXS15, as single class II GRX, within the mitochondria. We thus examined the distinct roles of the two main class I and class II GRXs, revealing key structural differences that determine their oxidoreductase and cluster-transferring functions. Specifically, we biochemically characterised GRXS15, to understand whether it can have catalytic activity besides its function in Fe–S cluster transfer. <br /> With a genetic approach, we then investigated the physiological consequences of reduced GRXS15 activity <em>in planta</em>. Exploiting <em>grxs15</em> knockdown mutants and partially complemented lines, we show that the GRXS15 diminished activity mainly affects the function of lipoyl synthase (LIP1). LIP1 has a low copy number per mitochondrion and requires a constant flux of [4Fe–4S] clusters for its catalytic activity. By knocking out aconitase 3 – the most abundant mitochondrial [4Fe–4S]-dependent enzyme – we observed the suppression of the dwarfism in the <em>grxs15</em> mutants. Similarly, we obtained analogous results by overexpression of LIP1. With these experiments, we demonstrate that LIP1 is not able to compete with other enzymes for receiving [4Fe–4S] clusters when the upstream delivery system is impaired. <br /> Unexpectedly, the overexpression of LIP1 was deleterious for wild-type plants. After having characterised this novel dwarf mutant we demonstrated that overexpression of LIP1 leads to the release of toxic amounts of sulfide, causing poisoning of the mitochondrial electron transport chain. Strikingly, we showed that this toxic effect can be alleviated by overexpression of <em>O</em>-acetylserine-(thiol)-lyase C, a component of the cysteine biosynthesis complex (CSC). We thus provide compelling evidence that the CSC acts as a sulfide detoxification system in the mitochondrion. Overall, this thesis contributes to refining the general overview of the entire pathway of mitochondrial Fe–S clusters, from assembly to consumption.