The iron-sulfur cluster assembly (ISC) protein Iba57 executes a tetrahydrofolate-independent function in mitochondrial [4Fe-4S] protein maturation.

Mitochondria harbor the bacteria-inherited iron-sulfur cluster assembly (ISC) machinery to generate [2Fe-2S; iron-sulfur (Fe-S)] and [4Fe-4S] proteins. In yeast, assembly of [4Fe-4S] proteins specifically involves the ISC proteins Isa1, Isa2, Iba57, Bol3, and Nfu1. Functional defects in their human equivalents cause the multiple mitochondrial dysfunction syndromes, severe disorders with a broad clinical spectrum. The bacterial Iba57 ancestor YgfZ was described to require tetrahydrofolate (THF) for its function in the maturation of selected [4Fe-4S] proteins. Both YgfZ and Iba57 are structurally related to an enzyme family catalyzing THF-dependent one-carbon transfer reactions including GcvT of the glycine cleavage system. On this basis, a universally conserved folate requirement in ISC-dependent [4Fe-4S] protein biogenesis was proposed. To test this idea for mitochondrial Iba57, we performed genetic and biochemical studies in Saccharomyces cerevisiae, and we solved the crystal structure of Iba57 from the thermophilic fungus Chaetomium thermophilum. We provide three lines of evidence for the THF independence of the Iba57-catalyzed [4Fe-4S] protein assembly pathway. First, yeast mutants lacking folate show no defect in mitochondrial [4Fe-4S] protein maturation. Second, the 3D structure of Iba57 lacks many of the side-chain contacts to THF as defined in GcvT, and the THF-binding pocket is constricted. Third, mutations in conserved Iba57 residues that are essential for THF-dependent catalysis in GcvT do not impair Iba57 function in vivo, in contrast to an exchange of the invariant, surface-exposed cysteine residue. We conclude that mitochondrial Iba57, despite structural similarities to both YgfZ and THF-binding proteins, does not utilize folate for its function.

Mitochondrial [4Fe-4S] protein assembly involves reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 by electron flow from ferredoxin FDX2.

The essential process of iron-sulfur (Fe/S) cluster assembly (ISC) in mitochondria occurs in three major phases. First, [2Fe-2S] clusters are synthesized on the scaffold protein ISCU2; second, these clusters are transferred to the monothiol glutaredoxin GLRX5 by an Hsp70 system followed by insertion into [2Fe-2S] apoproteins; third, [4Fe-4S] clusters are formed involving the ISC proteins ISCA1-ISCA2-IBA57 followed by target-specific apoprotein insertion. The third phase is poorly characterized biochemically, because previous in vitro assembly reactions involved artificial reductants and lacked at least one of the in vivo-identified ISC components. Here, we reconstituted the maturation of mitochondrial [4Fe-4S] aconitase without artificial reductants and verified the [2Fe-2S]-containing GLRX5 as cluster donor. The process required all components known from in vivo studies (i.e., ISCA1-ISCA2-IBA57), yet surprisingly also depended on mitochondrial ferredoxin FDX2 and its NADPH-coupled reductase FDXR. Electrons from FDX2 catalyze the reductive [2Fe-2S] cluster fusion on ISCA1-ISCA2 in an IBA57-dependent fashion. This previously unidentified electron transfer was occluded during previous in vivo studies due to the earlier FDX2 requirement for [2Fe-2S] cluster synthesis on ISCU2. The FDX2 function is specific, because neither FDX1, a mitochondrial ferredoxin involved in steroid production, nor other cellular reducing systems, supported maturation. In contrast to ISC factor-assisted [4Fe-4S] protein assembly, [2Fe-2S] cluster transfer from GLRX5 to [2Fe-2S] apoproteins occurred spontaneously within seconds, clearly distinguishing the mechanisms of [2Fe-2S] and [4Fe-4S] protein maturation. Our study defines the physiologically relevant mechanistic action of late-acting ISC factors in mitochondrial [4Fe-4S] cluster synthesis, trafficking, and apoprotein insertion.