Mechanistic characterization of ISC assembly factors involved in the maturation of mitochondrial [2Fe-2S] and [4Fe-4S] proteins

Abstract

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…