For iron-sulfur (Fe-S) cluster synthesis in mitochondria, the sulfur is derived from the amino acid cysteine by the cysteine desulfurase activity of Nfs1. The enzyme binds the substrate cysteine in the pyridoxal phosphate-containing site, and a persulfide is formed on the active site cysteine in a manner depending on the accessory protein Isd11. The persulfide is then transferred to the scaffold Isu, where it combines with iron to form the Fe-S cluster intermediate. Frataxin is implicated in the process, although it is unclear where and how, and deficiency causes Friedreich ataxia. Using purified proteins and isolated mitochondria, we show here that the yeast frataxin homolog (Yfh1) directly and specifically stimulates cysteine binding to Nfs1 by exposing substrate-binding sites. This novel function of frataxin does not require iron, Isu1, or Isd11. Once bound to Nfs1, the substrate cysteine is the source of the Nfs1 persulfide, but this step is independent of frataxin and strictly dependent on Isd11. Recently, a point mutation in Isu1 was found to bypass many frataxin functions. The data presented here show that the Isu1 suppressor mimics the frataxin effects on Nfs1, explaining the bypassing activity. We propose a regulatory mechanism for the Nfs1 persulfide-forming activity. Specifically, at least two separate conformational changes must occur in the enzyme for optimum activity as follows: one is mediated by frataxin interaction that exposes the “buried” substrate-binding sites, and the other is mediated by Isd11 interaction that brings the bound substrate cysteine and the active site cysteine in proximity for persulfide formation.

Iron–sulfur clusters are essential cofactors of proteins. In eukaryotes, iron–sulfur cluster biogenesis requires a mitochondrial iron–sulfur cluster machinery (ISC) and a cytoplasmic iron–sulfur protein assembly machinery (CIA). Here we used mitochondria and cytoplasm isolated from yeast cells, and [³⁵S]cysteine to detect cytoplasmic Fe–³⁵S cluster assembly on a purified apoprotein substrate. We showed that mitochondria generate an intermediate, called (Fe–S)_int, needed for cytoplasmic iron–sulfur cluster assembly. The mitochondrial biosynthesis of (Fe–S)_int required ISC components such as Nfs1 cysteine desulfurase, Isu1/2 scaffold, and Ssq1 chaperone. Mitochondria then exported (Fe–S)_int via the Atm1 transporter in the inner membrane, and we detected (Fe–S)_int in active form. When (Fe–S)_int was added to cytoplasm, CIA utilized it for iron–sulfur cluster assembly without any further help from the mitochondria. We found that both iron and sulfur for cytoplasmic iron–sulfur cluster assembly originate from the mitochondria, revealing a surprising and novel mitochondrial role. Mitochondrial (Fe–S)_int export was most efficient in the presence of cytoplasm containing an apoprotein substrate, suggesting that mitochondria respond to the cytoplasmic demand for iron–sulfur cluster synthesis. Of note, the (Fe–S)_int is distinct from the sulfur intermediate called S_int, which is also made and exported by mitochondria but is instead used for cytoplasmic tRNA thiolation. In summary, our findings establish a direct and vital role of mitochondria in cytoplasmic iron–sulfur cluster assembly in yeast cells.

Iron-sulfur (Fe-S) clusters are essential cofactors, and mitochondria contain several Fe-S proteins, including the [4Fe-4S] protein aconitase and the [2Fe-2S] protein ferredoxin. Fe-S cluster assembly of these proteins occurs within mitochondria. Although considerable data exist for yeast mitochondria, this biosynthetic process has never been directly demonstrated in mammalian mitochondria. Using [³⁵S]cysteine as the source of sulfur, here we show that mitochondria isolated from Cath.A-derived cells, a murine neuronal cell line, can synthesize and insert new Fe-³⁵S clusters into aconitase and ferredoxins. The process requires GTP, NADH, ATP, and iron, and hydrolysis of both GTP and ATP is necessary. Importantly, we have identified the ³⁵S-labeled persulfide on the NFS1 cysteine desulfurase as a genuine intermediate en route to Fe-S cluster synthesis. In physiological settings, the persulfide sulfur is released from NFS1 and transferred to a scaffold protein, where it combines with iron to form an Fe-S cluster intermediate. We found that the release of persulfide sulfur from NFS1 requires iron, showing that the use of iron and sulfur for the synthesis of Fe-S cluster intermediates is a highly coordinated process. The release of persulfide sulfur also requires GTP and NADH, probably mediated by a GTPase and a reductase, respectively. ATP, a cofactor for a multifunctional Hsp70 chaperone, is not required at this step. The experimental system described here may help to define the biochemical basis of diseases that are associated with impaired Fe-S cluster biogenesis in mitochondria, such as Friedreich ataxia.Fe-S cluster assembly in mitochondria involves generation of an activated form of sulfur called persulfide. Results A novel experimental system tracks new Fe-S cluster synthesis in isolated mammalian mitochondria. Conclusion The use of persulfide sulfur and iron for Fe-S cluster biogenesis is tightly coordinated by processes requiring GTP, NADH, and ATP. Significance These cofactor targets can now be defined.

Iron-sulfur (Fe-S) protein biogenesis in eukaryotes is mediated by two different machineries-one in the mitochondria and another in the cytoplasm. Glutaredoxin 5 (Grx5) is a component of the mitochondrial iron-sulfur cluster machinery. Here, we define the roles of Grx5 in maintaining overall mitochondrial/cellular Fe-S protein biogenesis, utilizing mitochondria and cytoplasm isolated from Saccharomyces cerevisiae cells. We previously demonstrated that isolated wild-type (WT) mitochondria themselves can synthesize new Fe-S clusters, but isolated WT cytoplasm alone cannot do so unless it is mixed with WT mitochondria. WT mitochondria generate an intermediate, called (Fe-S)_int, that is exported to the cytoplasm and utilized for cytoplasmic Fe-S cluster assembly. We here show that mitochondria lacking endogenous Grx5 (Grx5↓) failed to synthesize Fe-S clusters for proteins within the organelle. Similarly, Grx5↓ mitochondria were unable to synthesize (Fe-S)_int, as judged by their inability to promote Fe-S cluster biosynthesis in WT cytoplasm. Most importantly, purified Grx5 precursor protein, imported into isolated Grx5↓ mitochondria, rescued these Fe-S cluster synthesis/trafficking defects. Notably, mitochondria lacking immediate downstream components of the mitochondrial iron-sulfur cluster machinery (Isa1 or Isa2) could synthesize [2Fe-2S] but not [4Fe-4S] clusters within the organelle. Isa1↓ (or Isa2↓) mitochondria could still support Fe-S cluster biosynthesis in WT cytoplasm. These results provide evidence for Grx5 serving as a central hub for Fe-S cluster intermediate trafficking within mitochondria and export to the cytoplasm. Grx5 is conserved from yeast to humans, and deficiency or mutation causes fatal human diseases. Data as presented here will be informative for human physiology.