Molecular mechanisms of mitochondrial de novo [2Fe-2S] cluster formation and lipoyl biosynthesis
Iron-sulfur (Fe/S) clusters are small inorganic protein cofactors found in almost all known organisms. They enable various protein functions including electron transfer and catalysis and are integral to numerous essential biological processes like cellular respi-ration, translation, and DNA synthesi...
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|Summary:||Iron-sulfur (Fe/S) clusters are small inorganic protein cofactors found in almost all known organisms. They enable various protein functions including electron transfer and catalysis and are integral to numerous essential biological processes like cellular respi-ration, translation, and DNA synthesis and repair. Fe/S clusters typically exhibit simple structures, with the rhombic [2Fe-2S]- and cubic [4Fe-4S]-types being the most com-mon. Nevertheless, complex protein machineries are required for their biosynthesis and insertion into target apo-proteins. Mitochondrial Fe/S protein biogenesis requires the Fe/S cluster assembly (ISC) machinery consisting of up to 18 different proteins. The early ISC machinery assembles the [2Fe-2S] clusters de novo, and the late ISC ma-chinery uses these clusters to produce and insert [4Fe-4S] clusters.
Despite the functions of many proteins of the ISC machinery being well characterized, the molecular mechanisms underlying mitochondrial Fe/S protein biogenesis, in particu-lar de novo [2Fe-2S] cluster assembly, are not fully understood. The overarching aim of the first of the two projects in this work was to decipher at the molecular level how Fe and S are assembled on the scaffold protein ISCU2 to form [2Fe-2S] clusters de novo. In this process, one Fe ion and one persulfide moiety are delivered in a stepwise man-ner to the ISCU2 assembly site, which exhibits five conserved residues (Cys69, Asp71, Cys95, His137 and Cys138) believed to be critical for assembly. Efficient persulfidation of one of the three conserved ISCU2 Cys residues requires the heterodimeric cysteine desulfurase complex NFS1-ISD11-ACP (termed (NIA)2) to bind to both ISCU2 (U) and FXN (X), forming (NIAUX)2. The ISCU2-bound persulfide is reduced to sulfide via elec-tron flow from the ferredoxin FDX2, and finally dimerization of two [1Fe-1S] ISCU2 units enables [2Fe-2S] cluster formation. It was shown in this work that NFS1 persulfi-dates ISCU2 Cys138 efficiently and with high specificity, and no detectable sulfur relay via other ISCU2 Cys residues was observed. Importantly, ISCU2 had to be preloaded with one Fe(II) ion to enable physiologically relevant persulfidation. Furthermore, the ISCU2 residues Cys69, Cys95, Cys138 and likely Asp71 were identified as ligands of the mature [2Fe-2S] cluster.
A combined structural, spectroscopic and biochemical approach revealed the hitherto ill-defined Fe coordination by ISCU2 at various intermediate stages of [2Fe-2S] cluster synthesis. Initially, Fe(II) is coordinated by free ISCU2 in a tetrahedral fashion (via Cys69, Asp71, Cys95 and His137). Binding of ISCU2 to (NIA)2 was found to induce an equilibrium between the tetrahedral and a distinct octahedral coordination (via Asp71, Cys95, Cys138 and water ligands). The tetrahedral coordination was favored in (Fe-NIAU)2, but the binding of FXN, leading to the formation of (Fe-NIAUX)2, shifted the equilibrium towards the octahedral species. Specific intermolecular interactions be-tween FXN and ISCU2 assembly site residues support the formation of the octahedral species and are required for efficient [2Fe-2S] cluster synthesis. Furthermore, the 3D structure of the (Fe-NIAUX)2 complex with persulfidated ISCU2 Cys138 was obtained by electron cryo-microscopy at 2.4 Å resolution, which is the first (NIAUX)2 structure resolved below 3 Å. The Cys138 persulfide moiety participated in an octahedral Fe co-ordination similar to that in non-persulfidated complexes. Together, the aforementioned studies enabled the delineation of a detailed mechanistic route to physiological ISCU2 persulfidation as a decisive intermediate of [2Fe-2S] cluster synthesis.
The second project of this work focused on the function of human lipoyl synthase (LI-AS), a mitochondrial radical S-adenosyl methionine (SAM) [4Fe-4S] enzyme. Lipoyl is a cofactor of α-ketoacid dehydrogenases as well as the glycine cleavage system and thus integral to mitochondrial carbon metabolism. LIAS possesses a catalytic and an auxiliary [4Fe-4S] cluster. The catalytic cluster receives electrons to initiate a radical SAM-based reaction mechanism in which two sulfur atoms from the auxiliary cluster are incorporated into an octanoyl substrate. Despite extensive characterisation of the molecular mechanism of lipoylation, the physiological electron donor for the catalytic cluster of human LIAS has remained unknown. To address this issue, an in vitro assay closely mimicking human lipoyl biosynthesis was developed. It was found that only the mitochondrial ferredoxin FDX1, but not the structurally similar FDX2, serves as an effi-cient electron donor for LIAS catalysis. This finding was corroborated by AlphaFold-based in silico analyses of LIAS-FDX interactions. FDX1 supported in vitro lipoylation much more efficiently than the commonly employed artificial reductant dithionite. The high specificity of lipoylation for FDX1 was found to be connected to the C-terminus, because removal of the conserved FDX2 C-terminus largely enhanced residual FDX2 function in lipoylation. The in vitro lipoylation assay was also employed to investigate the toxic effect of elesclomol (Ele), an anticancer agent and copper ionophore. It was shown that both Cu and the Ele:Cu complex, but not Ele alone, inhibit lipoylation, thus identifying the major cellular target of Ele toxicity.
In summary, this work structurally defined the cooperative action of five ISCU2 resi-dues critical for consecutive states of de novo [2Fe-2S] cluster synthesis, and thereby provides valuable insights into the molecular dynamics of this process. Furthermore, the work contributes towards a better understanding of human lipoyl biosynthesis and the highly distinct functions of the two human FDXs. FDX1, in addition to its long-known role in steroidogenesis, was revealed as the physiological electron donor for LIAS catal-ysis.|
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