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Iron sulfur (Fe/S) proteins occur in all domains of life, and they fulfil essential functions in electron transfer, catalysis, and regulation of gene expression. In (non-green) eukaryotes, two assembly systems are involved in the maturation of Fe/S proteins located in mitochondria, cytosol and nucleus: The cytosolic iron-sulfur protein assembly (CIA) machinery with its 11 protein factors and the mitochondrial iron sulfur cluster (ISC) assembly machinery, which is composed of 17 proteins and participates in the maturation of all cellular Fe/S proteins. Biogenesis of Fe/S-clusters in the ISC system involves the de novo synthesis of a [2Fe-2S] cluster on the scaffold protein ISCU, and its subsequent transfer to the monothiol glutaredoxin GLRX5 by the means of a dedicated Hsp70 chaperon system. The transiently bound [2Fe-2S] clusters on GLRX5 can be transferred to [2Fe-2S] target apoproteins or are converted to a [4Fe-4S] cluster. Based on in vivo findings the human ISC proteins ISCA1, ISCA2 and IBA57 are essential for the formation of a [4Fe-4S] cluster. Moreover, specialized ISC proteins are needed for the insertion of [4Fe-4S] clusters into target proteins. The ISC targeting factors NFU1 assists Fe/S-cluster insertion into dedicated apoproteins like aconitase (ACO2) by transiently binding a [4Fe-4S] cluster. Two other proteins, BOLA1 and BOLA3, are supposed to be additional specific ISC targeting factors. Although the synthesis of [2Fe-2S] clusters on ISCU is becoming well understood, the formation of [4Fe-4S] clusters and the mechanism of transfer of [2Fe-2S] or [4Fe-4S] clusters to recipient proteins is largely unknown. For the eukaryotic systems neither [2Fe-2S] nor [4Fe-4S] cluster transfer experiments in vitro were performed under physiological conditions, because all previous experiments contained the artificial thiol-specific reductant dithiothreitol (DTT). The often essential role of DTT in these in vitro systems is not understood so far and it is not clear, what compound might replace DTT in vivo.
In this work, substrate-specific [2Fe-2S] and [4Fe-4S] cluster transfer reactions were established in the presence of the physiological cellular thiol-reducing system composed of the dithiol glutaredoxin Grx1 and glutathione (GSH). In the presence of this thiol redox system, Fe/S cluster transfer reactions involving the mitochondrial ISC transfer factor GLRX5 displayed substrate specificity and unprecedented speed: Transfer of a [2Fe-2S] cluster from human GLRX5 to human ferredoxin (FDX1), its natural substrate, was efficiently completed in less than 15 seconds. The [4Fe-4S] ISC transfer factor NFU1 also efficiently matured [4Fe-4S] proteins (ACO2, Leu1) in the presence of Grx1/GSH. In contrast, the direct transfer of a [2Fe-2S]-cluster from GLRX5 to a [4Fe-4S] proteins failed in the presence of Grx1 and GSH, indicating that [2Fe-2S]2+ clusters are not reductively coupled to a [4Fe-4S]2+ cluster by Grx1/GSH. However, in the presence of the human ISC proteins ISCA1, ISCA2, IBA57 and an electron transfer chain (FDX2/FDXR/NADPH) the conversion of [2Fe-2S]-clusters into [4Fe-4S]-cluster and its insertion into ACO2 was observed in the presence of Grx1/GSH. In this reaction, the transiently bound [2Fe-2S] cluster on GLRX5 functions as a Fe/S donor. Taken together, in the first part of the work in vitro Fe/S transfer systems were developed that faithfully mimic a [2Fe-2S] and [4Fe-4S] cluster transfer reactions without artificial additives. While the transfer of [2Fe-2S] cluster from GLRX5 was spontaneous, the formation and insertion of [4Fe-4S] clusters on aconitase depended on all ISC components known to be required in vivo for [4Fe-4S] cluster synthesis and on an electron transfer chain. These findings strongly suggest that the transfer reactions closely resemble the in vivo situation.
Two proteins of the BOLA family, BOLA1 and BOLA3, likely act as specific ISC targeting factors. Patients with a functional loss of BOLA3 suffer from a multiple mitochondrial dysfunction syndrome 2 (MMDS2). This patient phenotype was similar to patients who had mutations in the gene for the specific ISC targeting factor NFU1 (MMDS1). In vivo analysis characterized the yeast Bol proteins as specific mitochondrial ISC factors that facilitate [4Fe-4S] cluster insertion into a subset of mitochondrial proteins such as lipoate synthase and succinate dehydrogenase. The yeast Bol proteins perform largely overlapping functions.
Here, it is shown by in vitro methods (MicroScale Thermophoresis, analytical gel filtration, UV/Vis- and CD-spectroscopy) that human BOLA1 and BOLA3 form heterodimeric complexes with GLRX5. BOLA1 interacts with both the apo- and holo-form of GLRX5. Compared to a homodimeric holoGLRX5-GLRX5 complex, the heterodimeric holoGLRX5-BOLA1-complex displays altered CD-spectroscopic features, the [2Fe-2S] cluster was more stable, protected against reduction, and could not be transferred to [2Fe-2S] target proteins. To determine the coordination ligands of the [2Fe-2S] cluster in a holoGLRX5-BOLA1 complex, conserved histidine residues (H102, H67, H58) were replaced by alanine. A GLRX5-BOLA1H102A complex was stable against reduction and displayed similar CD-spectroscopic features as the native GLRX5-BOLA1 complex. Histidine residue H102 is essential in vivo, but apparently not needed for Fe/S cluster binding in vitro. In contrast, the Fe/S cluster on a GLRX5-BOLA1mut complex (mut: H58A, H67A, H102/67/58A) was reductively labile and displayed altered CD-spectroscopic features. When all conserved histidine residues were replaced by alanine, Fe/S cluster binding was still detectable suggesting that GSH can stabilize the Fe/S cluster in a GLRX5-BOLA1 complex. BOLA3 showed a different behavior. It formed a dimeric complex with the apo form of GLRX5, but formation of the holo complex required the presence of GSH. This holoGLRX5-BOLA3 complex bound a [2Fe-2S] cluster in labile fashion. The cluster was not protected against reduction and Fe/S cluster transfer rates of the homodimeric GLRX5 and the GLRX5-BOLA3 complex were nearly identical.
Taken together, the two GLRX5-BOLA complexes displayed distinct biochemical characteristics in vitro suggesting that the human BOLA proteins perform differential functions in vivo.