Mechanisms of iron-sulfur protein maturation in mitochondria, cytosol and nucleus of eukaryotes

Iron-sulfur (Fe/S) clusters are important cofactors of numerous proteins involved in electron transfer, metabolic and regulatory processes. In eukaryotic cells, known Fe/S proteins are located within mitochondria, the nucleus and the cytosol. Over the past years the molecular basis of Fe/S cluster synthesis and incorporation into apoproteins in a living cell has started to become elucidated. Biogenesis of these simple inorganic cofactors is surprisingly complex and, in eukaryotes such as Saccharomyces cerevisiae, is accomplished by three distinct proteinaceous machineries. The "iron-sulfur cluster (ISC) assembly machinery" of mitochondria was inherited from the bacterial ancestor of mitochondria. ISC components are conserved in eukaryotes from yeast to man. The key principle of biosynthesis is the assembly of the Fe/S cluster on a scaffold protein before it is transferred to target apoproteins. Cytosolic and nuclear Fe/S protein maturation also requires the function of the mitochondrial ISC assembly system. It is believed that mitochondria contribute a still unknown compound to biogenesis outside the organelle. This compound is exported by the mitochondrial "ISC export machinery" and utilised by the "cytosolic iron-sulfur protein assembly (CIA) machinery". Components of these two latter systems are also highly conserved in eukaryotes. Defects in the mitochondrial ISC assembly and export systems, but not in the CIA machinery have a strong impact on cellular iron uptake and intracellular iron distribution showing that mitochondria are crucial for both cellular Fe/S protein assembly and iron homeostasis.     

Frataxin-mediated iron delivery to ferrochelatase in the final step of heme biosynthesis

Human ferrochelatase, a mitochondrial membrane-associated protein, catalyzes the terminal step of heme biosynthesis by insertion of ferrous iron into protoporphyrin IX. The recently solved x-ray structure of human ferrochelatase identifies a potential binding site for an iron donor protein on the matrix side of the homodimer. Herein we demonstrate Hs holofrataxin to be a high affinity iron binding partner for Hs ferrochelatase that is capable of both delivering iron to ferrochelatase and mediating the terminal step in mitochondrial heme biosynthesis. A general regulatory mechanism for mitochondrial iron metabolism is described that defines frataxin involvement in both heme and iron-sulfur cluster biosyntheses. In essence, the distinct binding affinities of holofrataxin to the target proteins, ferrochelatase (heme synthesis) and ISU (iron-sulfur cluster synthesis), allows discrimination between the two major iron-dependent pathways and facilitates targeted heme biosynthesis following down-regulation of frataxin.     

The human mitochondrial ISCA1, ISCA2, and IBA57 proteins are required for [4Fe-4S] protein maturation

Members of the bacterial and mitochondrial iron-sulfur cluster (ISC) assembly machinery include the so-called A-type ISC proteins, which support the assembly of a subset of Fe/S apoproteins. The human genome encodes two A-type proteins, termed ISCA1 and ISCA2, which are related to Saccharomyces cerevisiae Isa1 and Isa2, respectively. An additional protein, Iba57, physically interacts with Isa1 and Isa2 in yeast. To test the cellular role of human ISCA1, ISCA2, and IBA57, HeLa cells were depleted for any of these proteins by RNA interference technology. Depleted cells contained massively swollen and enlarged mitochondria that were virtually devoid of cristae membranes, demonstrating the importance of these proteins for mitochondrial biogenesis. The activities of mitochondrial [4Fe-4S] proteins, including aconitase, respiratory complex I, and lipoic acid synthase, were diminished following depletion of the three proteins. In contrast, the mitochondrial [2Fe-2S] enzyme ferrochelatase and cellular heme content were unaffected. We further provide evidence against a localization and direct Fe/S protein maturation function of ISCA1 and ISCA2 in the cytosol. Taken together, our data suggest that ISCA1, ISCA2, and IBA57 are specifically involved in the maturation of mitochondrial [4Fe-4S] proteins functioning late in the ISC assembly pathway.     

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells

Mutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron-sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron-sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron-sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models.     

Mechanism of iron transport to the site of heme synthesis inside yeast mitochondria.

The import of metals, iron in particular, into mitochondria is poorly understood. Iron in mitochondria is required for the biosynthesis of heme and various iron-sulfur proteins. We have developed an in vitro assay to follow the uptake of iron into isolated yeast mitochondria. By measuring the incorporation of iron into porphyrin by ferrochelatase in the matrix, we were able to define the mechanism of iron import. Iron uptake is driven energetically by a membrane potential across the inner membrane but does not require ATP. Only reduced iron is functional in generating heme. Iron cannot be preloaded in the mitochondrial matrix but rather has to be transported across the inner membrane simultaneously with the synthesis of heme, suggesting that ferrochelatase receives iron directly from the inner membrane. Transport of iron is inhibited by manganese but not by zinc, nickel, and copper ions, explaining why in vivo these ions are not incorporated into porphyrin. The inner membrane proteins Mmt1p and Mmt2p proposed to be involved in mitochondrial iron movement are not required for the supply of ferrochelatase with iron. Iron transport can be reconstituted efficiently in a membrane potential-dependent fashion in proteoliposomes that were formed from a detergent extract of mitochondria. Our biochemical analysis of iron import into yeast mitochondria provides the basis for the identification of components involved in transport.     

线粒体铁代谢与人类疾病的研究进展

线粒体铁代谢的研究主要包括两个方面：铁在胞质和线粒体之间的转运和调控;铁硫簇和血红素在线粒体内的合成与转运。目前认为线粒体铁的转入主要是与mitoferrinl／2（MFRNl和MFRN2）和ABCBl0有关,运出可能与ABCB6和／或ABCB7有关,转运和调控的具体机制不是很清楚,推测与某种含有铁硫簇的信号分子有关。哺乳动物铁硫簇的合成可以发生在胞质和线粒体内,但以线粒体为主;真核生物中与铁硫簇合成相关的蛋白达二十多种,其中FXN、ISCS、ISDll和ISCU及其同系物被认为是核心组分。血红素的合成起始和终止发生在线粒体内,终止步骤为亚铁螯合酶将铁插入原卟啉IX,该酶活性又依赖于铁硫簇。因此,铁硫簇的合成与调控是线粒体铁代谢的核心,也是整个细胞铁运作的核心。本文主要围绕线粒体铁代谢特别是铁硫簇的合成异常引起的疾病做一简单的综述。     

Biogenesis of iron-sulfur proteins in eukaryotes: a novel task of mitochondria that is inherited from bacteria

Fe/S clusters are co-factors of numerous proteins with important functions in metabolism, electron transport and regulation of gene expression. Presumably, Fe/S proteins have occurred early in evolution and are present in cells of virtually all species. Biosynthesis of these proteins is a complex process involving numerous components. In mitochondria, this process is accomplished by the so-called ISC (iron-sulfur cluster assembly) machinery which is derived from the bacterial ancestor of the organelles and is conserved from lower to higher eukaryotes. The mitochondrial ISC machinery is responsible for biogenesis iron-sulfur proteins both within and outside the organelle. Maturation of the latter proteins involves the ABC transporter Atm1p which presumably exports iron-sulfur clusters from the organelle. This review summarizes recent developments in our understanding of the biogenesis of iron-sulfur proteins both within bacteria and eukaryotes.     

Depletion of thiol reducing capacity impairs cytosolic but not mitochondrial iron-sulfur protein assembly machineries

Iron‑sulfur (Fe/S) clusters are versatile inorganic cofactors that play central roles in essential cellular functions, from respiration to genome stability. >30 proteins involved in Fe/S protein biogenesis in eukaryotes are known, many of which bind clusters via cysteine residues. This opens up the possibility that the thiol-reducing glutaredoxin and thioredoxin systems are required at both the Fe/S biogenesis and target protein level to counteract thiol oxidation. To address the possible interplay of thiol redox chemistry and Fe/S protein biogenesis, we have characterized the status of the mitochondrial (ISC) and cytosolic (CIA) Fe/S protein assembly machineries in Saccharomyces cerevisiae mutants in which the three partially redundant glutathione (Glr1) and thioredoxin (Trr1 and Trr2) oxidoreductases have been inactivated in either mitochondria, cytosol, or both compartments. Cells devoid of mitochondrial oxidoreductases maintained a functional mitochondrial ISC machinery and showed no altered iron homeostasis despite a non-functional complex II of the respiratory chain due to redox-specific defects. In cells that lack either cytosolic or total cellular thiol reducing capacity, both the ISC system and iron homeostasis were normal, yet cytosolic and nuclear Fe/S target proteins were not matured. This dysfunction could be attributed to a failure in the assembly of [4Fe‑4S] clusters in the CIA factor Nar1, even though Nar1 maintained robust protein levels and stable interactions with later-acting CIA components. Overall, our analysis has uncovered a hitherto unknown thiol-dependence of the CIA machinery and has demonstrated the surprisingly varying sensitivity of Fe/S proteins to thiol oxidation.     

The yeast scaffold proteins Isu1p and Isu2p are required inside mitochondria for maturation of cytosolic Fe/S proteins

Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.     

A third bacterial system for the assembly of iron-sulfur clusters with homologs in archaea and plastids

The assembly of iron-sulfur (Fe-S) clusters is mediated by complex machinery. In several proteobacteria, this process involves ISC (Fe-S cluster assembly) machinery composed of at least six components also conserved in mitochondria from lower to higher eukaryotes. In nitrogen-fixing bacteria, another system, termed NIF (nitrogen fixation), is required for the maturation of nitrogenase. Here we report the identification of a third system, designated the SUF machinery, the components of which are encoded in Escherichia coli by an unassigned operon, sufABCDSE. We have analyzed spontaneous pseudorevertants isolated from a mutant strain lacking all the components of the ISC machinery. The suppressor mutations in the revertants have been localized to the regulatory region of the suf operon; overexpression of this operon restores the growth phenotypes and activity of Fe-S proteins in mutant cells lacking ISC. Disruption of the suf operon alone does not cause any major defects, but synthetic lethality was observed when both the isc and suf operons were inactivated. These results indicate that proteins encoded by the suf operon participate in the ISC-independent minor pathway for the assembly of Fe-S clusters. The genes homologous to sufBC are present in a wide range of bacteria, Archaea, and plastids, suggesting that this type of system is almost ubiquitous in nature.     

Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins

Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron-sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.     

Frataxin, a conserved mitochondrial protein, in the hydrogenosome of Trichomonas vaginalis

Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical alpha-beta sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.     

Grx5 is a mitochondrial glutaredoxin required for the activity of iron/sulfur enzymes

Yeast cells contain a family of three monothiol glutaredoxins: Grx3, 4, and 5. Absence of Grx5 leads to constitutive oxidative damage, exacerbating that caused by external oxidants. Phenotypic defects associated with the absence of Grx5 are suppressed by overexpression of SSQ1 and ISA2, two genes involved in the synthesis and assembly of iron/sulfur clusters into proteins. Grx5 localizes at the mitochondrial matrix, like other proteins involved in the synthesis of these clusters, and the mature form lacks the first 29 amino acids of the translation product. Absence of Grx5 causes: 1) iron accumulation in the cell, which in turn could promote oxidative damage, and 2) inactivation of enzymes requiring iron/sulfur clusters for their activity. Reduction of iron levels in grx5 null mutants does not restore the activity of iron/sulfur enzymes, and cell growth defects are not suppressed in anaerobiosis or in the presence of disulfide reductants. Hence, Grx5 forms part of the mitochondrial machinery involved in the synthesis and assembly of iron/sulfur centers.     

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bäckström, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162–175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04±0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37°C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.     

Specialized function of yeast Isa1 and Isa2 proteins in the maturation of mitochondrial [4Fe-4S] proteins

Most eukaryotes contain iron-sulfur cluster (ISC) assembly proteins related to Saccharomyces cerevisiae Isa1 and Isa2. We show here that Isa1 but not Isa2 can be functionally replaced by the bacterial relatives IscA, SufA, and ErpA. The specific function of these "A-type" ISC proteins within the framework of mitochondrial and bacterial Fe/S protein biogenesis is still unresolved. In a comprehensive in vivo analysis, we show that S. cerevisiae Isa1 and Isa2 form a complex that is required for maturation of mitochondrial [4Fe-4S] proteins, including aconitase and homoaconitase. In contrast, Isa1-Isa2 were dispensable for the generation of mitochondrial [2Fe-2S] proteins and cytosolic [4Fe-4S] proteins. Targeting of bacterial [2Fe-2S] and [4Fe-4S] ferredoxins to yeast mitochondria further supported this specificity. Isa1 and Isa2 proteins are shown to bind iron in vivo, yet the Isa1-Isa2-bound iron was not needed as a donor for de novo assembly of the [2Fe-2S] cluster on the general Fe/S scaffold proteins Isu1-Isu2. Upon depletion of the ISC assembly factor Iba57, which specifically interacts with Isa1 and Isa2, or in the absence of the major mitochondrial [4Fe-4S] protein aconitase, iron accumulated on the Isa proteins. These results suggest that the iron bound to the Isa proteins is required for the de novo synthesis of [4Fe-4S] clusters in mitochondria and for their insertion into apoproteins in a reaction mediated by Iba57. Taken together, these findings define Isa1, Isa2, and Iba57 as a specialized, late-acting ISC assembly subsystem that is specifically dedicated to the maturation of mitochondrial [4Fe-4S] proteins.     

Biogenesis of iron-sulfur proteins in eukaryotes: components, mechanism and pathology

Iron-sulfur (Fe-S) clusters are ubiquitous co-factors of proteins that play an important role in metabolism, electron-transfer and regulation of gene expression. In eukaryotes mitochondria are the primary site of Fe-S cluster biogenesis. The organelles contain some ten proteins of the so-called iron-sulfur cluster (ISC) assembly machinery that is well-conserved in bacteria and eukaryotes. The ISC assembly machinery is responsible for biogenesis of Fe-S proteins within mitochondria. In addition, this machinery is involved in the maturation of extra-mitochondrial Fe-S proteins by cooperating with mitochondrial proteins with an exclusive function in this process. This review summarizes recent developments in our understanding of the biogenesis of cellular Fe-S proteins in eukaryotes. Particular emphasis is given to disorders in Fe-S protein biogenesis causing human disease.     

Assembly of human frataxin is a mechanism for detoxifying redox-active iron

Mitochondrial function depends on a continuous supply of iron to the iron-sulfur cluster (ISC) and heme biosynthetic pathways as well as on the ability to prevent iron-catalyzed oxidative damage. The mitochondrial protein frataxin plays a key role in these processes by a novel mechanism that remains to be fully elucidated. Recombinant yeast and human frataxin are able to self-associate in large molecular assemblies that bind and store iron as a ferrihydrite mineral. Moreover, either single monomers or polymers of human frataxin have been shown to serve as donors of Fe(II) to ISC scaffold proteins, oxidatively inactivated [3Fe-4S](+) aconitase, and ferrochelatase. These results suggest that frataxin can use different molecular forms to accomplish its functions. Here, stable monomeric and assembled forms of human frataxin purified from Escherichia coli have provided a tool for testing this hypothesis at the biochemical level. We show that human frataxin can enhance the availability of Fe(II) in monomeric or assembled form. However, the monomer is unable to prevent iron-catalyzed radical reactions and the formation of insoluble ferric iron oxides. In contrast, the assembled protein has ferroxidase activity and detoxifies redox-active iron by sequestering it in a protein-protected compartment.