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Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability

IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19*
  • Prasenjit Prasad Saha
    Footnotes
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka
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  • Shubhi Srivastava
    Footnotes
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka
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  • Praveen Kumar S. K.
    Footnotes
    Affiliations
    Department of Biochemistry, Karnatak University, Dharwad 580003, Karnataka, India
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  • Devanjan Sinha
    Footnotes
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka
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  • Patrick D'Silva
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Indian Institute of Science, Biological Sciences Building, Bangalore 560012, Karnataka, India. Tel.: 91-080-22932821; Fax: 91-080-23600814
    Affiliations
    Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka
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  • Author Footnotes
    * This work was supported in part by Council of Scientific and Industrial Research Grant CSIR 37(1534)/12/EMR-II, India (to P. D. S.), and Swarnajayanthi Fellowship from Department of Science and Technology (DST), Ministry of Science and Technology, India (to P. D. S.). The authors declare that they have no conflicts of interest with the contents of this article.
    1 Supported by Council of Scientific and Industrial Research, India for Senior Research fellowships.
    2 Supported by a DST, India INSPIRE fellowship.
    3 Supported by a Department of Biotechnology for Research Associate fellowship, India.
Open AccessPublished:September 04, 2015DOI:https://doi.org/10.1074/jbc.M115.678508
      Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

      Introduction

      Biological activity of proteins often requires the assistance of a non-protein chemical moiety called cofactors. Iron-sulfur (Fe-S) clusters are one of the most versatile and evolutionarily conserved cofactors that are present ubiquitously in all life forms ranging from ancient microorganisms to present day genera (
      • Craig E.A.
      • Marszalek J.
      A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers.
      ,
      • Craig E.A.
      • Voisine C.
      • Schilke B.
      Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae.
      ). Typically, these low molecular mass moieties bind to conserved sequence motifs of proteins essentially through cysteine or histidine residues (
      • Meyer J.
      Iron-sulfur protein folds, iron-sulfur chemistry, and evolution.
      ). More than 200 proteins require Fe-S clusters for their biological function, including members of the ETC complexes, enzymes, and several transcription factors to regulate gene expression. Moreover, Fe-S clusters can also act as sensors of iron and oxygen (
      • Lill R.
      • Dutkiewicz R.
      • Elsässer H.P.
      • Hausmann A.
      • Netz D.J.
      • Pierik A.J.
      • Stehling O.
      • Urzica E.
      • Mühlenhoff U.
      Mechanisms of iron-sulfur protein maturation in mitochondria, cytosol and nucleus of eukaryotes.
      ). Although the chemical structures of Fe-S clusters moieties appear simple, their biogenesis in a living cell is highly complex and coordinated process. Based on the number of iron and sulfur atoms, Fe-S clusters are assembled into two major types, [2Fe-2S] and [4Fe-4S] clusters. Besides, the presence of [3Fe-4S] clusters have also been reported in some Fe-S cluster proteins (
      • Beinert H.
      • Holm R.H.
      • Münck E.
      Iron-sulfur clusters: nature's modular, multipurpose structures.
      ).
      In higher eukaryotes, the majority of Fe-S cluster biogenesis takes place in mitochondria and consists of a series of complex events that can be categorized into three major steps. Initially, iron, sulfur, and electrons are transferred to a scaffold protein as raw materials for the biogenesis process. Second, assembling the Fe-S clusters in the scaffold protein. Finally, transfer of Fe-S clusters from the scaffold protein to a recipient apoprotein (
      • Craig E.A.
      • Marszalek J.
      A specialized mitochondrial molecular chaperone system: a role in formation of Fe/S centers.
      ). In humans, the process of sulfur transfer is assisted by the cysteine desulfurase protein NFS1 (IscS in bacteria and Nfs1 in yeast), whereas the precise source of iron is still elusive. However, the iron-binding protein frataxin (CyaY in bacteria and Yfh1 in yeast) is speculated to function as the putative iron donor by interacting with the sulfur donor and scaffold protein (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ). Ferredoxin reductase (Arh1 in yeast) transfers electrons from NADH to the scaffold protein via ferredoxin 2 (Fdx in bacteria and Yah1 in yeast). A conserved protein ISCU (IscU in bacteria and Isu1/2 in yeast) acts as a scaffold for the assembly of Fe-S cluster before its transfer to apoproteins. The transfer process is mediated by chaperone machinery comprising the mtHsp70/Mortalin (HscA in bacteria and Ssq1 in yeast) and the J-protein co-chaperone, HSCB (HscB in bacteria and Jac1 in yeast) along with monothiol glutaredoxin, AND GLRX5 (GrxD in bacteria and Grx5 in yeast) (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ). Based on the types of Fe-S clusters to be incorporated in the final recipient proteins, additional targeting factors are involved in the transfer process in higher eukaryotes (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ,
      • Maio N.
      • Rouault T.A.
      Iron-sulfur cluster biogenesis in mammalian cells: new insights into the molecular mechanisms of cluster delivery.
      ,
      • Stehling O.
      • Wilbrecht C.
      • Lill R.
      Mitochondrial iron-sulfur protein biogenesis and human disease.
      ). Although the functional importance of individual proteins in the biogenesis pathway has been reported, the molecular chemistry of Fe-S cluster assembly and transfer are not firmly established.
      The donation of the sulfur atoms is brought about by the NFS1 protein, which belongs to the aminotransferase subgroup of the pyridoxal 5-phosphate-dependent enzymes and is highly conserved from bacteria to humans (
      • Land T.
      • Rouault T.A.
      Targeting of a human iron-sulfur cluster assembly enzyme, nifs, to different subcellular compartments is regulated through alternative AUG utilization.
      ,
      • Biederbick A.
      • Stehling O.
      • Rösser R.
      • Niggemeyer B.
      • Nakai Y.
      • Elsässer H.P.
      • Lill R.
      Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation.
      ). NFS1 acts as a cysteine desulfurase to obtain sulfur from cysteine and forms alanine in this process. A conserved cysteine residue of NFS1 serves as a nucleophile to attack the γ-sulfhydryl group (γ-SH) of free cysteine amino acid and obtains the γ-SH to form a persulfide intermediate at the active site cysteine (
      • Mühlenhoff U.
      • Balk J.
      • Richhardt N.
      • Kaiser J.T.
      • Sipos K.
      • Kispal G.
      • Lill R.
      Functional characterization of the eukaryotic cysteine desulfurase Nfs1p from Saccharomyces cerevisiae.
      ,
      • Zheng L.
      • White R.H.
      • Cash V.L.
      • Dean D.R.
      Mechanism for the desulfurization of l-cysteine catalyzed by the nifS gene product.
      ,
      • Cupp-Vickery J.R.
      • Urbina H.
      • Vickery L.E.
      Crystal structure of IscS, a cysteine desulfurase from Escherichia coli.
      ,
      • Zheng L.
      • Dean D.R.
      Catalytic formation of a nitrogenase iron-sulfur cluster.
      ). The crystal structures of bacterial cysteine desulfurase protein highlights that it exists as a dimer and each monomer consists of two subdomains. The larger domain binds to the pyridoxal phosphate, and the smaller domain harbors the active site cysteine that transiently holds the sulfur as a persulfide released from cysteine (
      • Cupp-Vickery J.R.
      • Urbina H.
      • Vickery L.E.
      Crystal structure of IscS, a cysteine desulfurase from Escherichia coli.
      ,
      • Kaiser J.T.
      • Clausen T.
      • Bourenkow G.P.
      • Bartunik H.D.
      • Steinbacher S.
      • Huber R.
      Crystal structure of a NifS-like protein from Thermotoga maritima: implications for iron sulphur cluster assembly.
      ). In bacteria, the crystal structure of the NFS1 ortholog IscS shows that it forms a tight complex with IscU, thereby accelerating sulfur transfer from IscS to IscU (
      • Marinoni E.N.
      • de Oliveira J.S.
      • Nicolet Y.
      • Raulfs E.C.
      • Amara P.
      • Dean D.R.
      • Fontecilla-Camps J.C.
      (IscS-IscU)2 complex structures provide insights into Fe2S2 biogenesis and transfer.
      ,
      • Kato S.
      • Mihara H.
      • Kurihara T.
      • Takahashi Y.
      • Tokumoto U.
      • Yoshimura T.
      • Esaki N.
      Cys-328 of IscS and Cys-63 of IscU are the sites of disulfide bridge formation in a covalently bound IscS/IscU complex: implications for the mechanism of iron-sulfur cluster assembly.
      ). Biochemical and genetic analysis involving yeast Nfs1 showed that it stabilizes the scaffold protein Isu1 and modulates its level in different growth conditions (
      • Song J.Y.
      • Marszalek J.
      • Craig E.A.
      Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.
      ). Moreover, both Nfs1 and Jac1 exhibit binding to mutually exclusive sites in Isu1 (
      • Majewska J.
      • Ciesielski S.J.
      • Schilke B.
      • Kominek J.
      • Blenska A.
      • Delewski W.
      • Song J.Y.
      • Marszalek J.
      • Craig E.A.
      • Dutkiewicz R.
      Binding of the chaperone Jac1 protein and cysteine desulfurase Nfs1 to the iron-sulfur cluster scaffold Isu protein is mutually exclusive.
      ). However, the mechanism of sulfur transfer from Nfs1 to the scaffold protein is not yet fully established.
      In yeast, it has been reported that the Nfs1 protein remains in a complex with an 11-kDa protein named Isd11 (ISD11 in humans), which has no obvious sequence homologs in bacteria. Isd11 is an essential protein in yeast and depletion of ISD11 results in impairment of Fe-S cluster biogenesis (
      • Adam A.C.
      • Bornhövd C.
      • Prokisch H.
      • Neupert W.
      • Hell K.
      The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria.
      ,
      • Wiedemann N.
      • Urzica E.
      • Guiard B.
      • Müller H.
      • Lohaus C.
      • Meyer H.E.
      • Ryan M.T.
      • Meisinger C.
      • Mühlenhoff U.
      • Lill R.
      • Pfanner N.
      Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins.
      ). Additionally, it has been suggested that the Nfs1 protein is prone to aggregation and degradation in the absence of Isd11 (
      • Adam A.C.
      • Bornhövd C.
      • Prokisch H.
      • Neupert W.
      • Hell K.
      The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria.
      ). Although Isd11 is not required for desulfurase activity of Nfs1 in vitro, the Nfs1-Isd11 complex represents the functional sulfur donor, in vivo (
      • Wiedemann N.
      • Urzica E.
      • Guiard B.
      • Müller H.
      • Lohaus C.
      • Meyer H.E.
      • Ryan M.T.
      • Meisinger C.
      • Mühlenhoff U.
      • Lill R.
      • Pfanner N.
      Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins.
      ). In humans, the Isd11 ortholog ISD11 is believed to play a similar conserved role in Fe-S cluster biogenesis (
      • Shi Y.
      • Ghosh M.C.
      • Tong W.H.
      • Rouault T.A.
      Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis.
      ) and loss of ISD11 function results in a mitochondrial disorder termed as combined oxidative phosphorylation deficiency 19 (COXPD19)
      The abbreviations used are: COXPD19
      combined oxidative phosphorylation deficiency 19
      H2DCFDA
      2′,7′-dichlorodihydrofluorescein diacetate
      5-FOA
      5-fluoroorotic acid
      AAS
      atomic absorption spectroscopy
      NAO
      10-N-nonyl acridine orange
      Ni-NTA
      nickel-nitrilotriacetic acid
      ROS
      reactive oxygen species.
      (
      • Lim S.C.
      • Friemel M.
      • Marum J.E.
      • Tucker E.J.
      • Bruno D.L.
      • Riley L.G.
      • Christodoulou J.
      • Kirk E.P.
      • Boneh A.
      • DeGennaro C.M.
      • Springer M.
      • Mootha V.K.
      • Rouault T.A.
      • Leimkühler S.
      • Thorburn D.R.
      • Compton A.G.
      Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes.
      ).
      Recent studies on human ISD11 have shown that a homozygous mutation in the LYRM4 gene on chromosome 6p25, which codes for the ISD11 protein, converts a conserved arginine residue at position 68 into leucine (R68L) implicated in the mitochondrial genetic disorder COXPD19. This disorder is characterized by respiratory distress, hypotonia, gastroesophageal reflux, hepatomegaly, and severe lactic acidosis in the neonates. Studies on patient liver and muscle samples have demonstrated decreased activities of mitochondrial ETC complexes I–IV. Postmortem examination of COXPD19 patients had shown widened fiber size in the skeletal muscle, increased lipid content in muscle and liver along with the presence of abnormal mitochondria as detected through electron microscopic analysis. It is known that the R68L mutation in the ISD11 protein leads to development of COXPD19, although detailed molecular mechanisms associated with the cellular defects have not been elucidated.
      Because of the crucial importance of the ISD11 protein in Fe-S cluster biogenesis, we have delineated the role of indispensable ISD11 residues for mitochondrial function by utilizing yeast and cell lines as model systems. In this report, we have mapped key residues on the ISD11 protein that are essential for NFS1 interaction to maintain its levels by forming a stable subcomplex that is critical for Fe-S cluster biogenesis. Additionally, our study uncovers the cellular defects associated with the R68L ISD11 mutation, thus revealing biochemical insights into COXPD19 progression. Our findings highlight that the R68L mutation results in impaired Fe-S cluster biogenesis, elevated mitochondrial iron, and oxidative stress, which contribute significantly toward the development of COXPD19.

      Discussion

      In the present study, we have delineated the importance of the ISD11 protein as a part of the sulfur-transfer protein complex, NFS1-ISD11 in Fe-S cluster biogenesis pathway. Our results indicate that the ISD11 protein interacts with the cysteine desulfurase protein NFS1 and prevents its self-aggregation, thus assisting a functional Fe-S cluster biogenesis process in the mitochondrial compartment. Importantly, this is a first report that maps the crucial residues of ISD11 protein that involves in interaction with NFS1. Furthermore, this report delivers mechanistic insights on the possible explanation of COXPD19 development and progression as a result of R68L mutation in ISD11 protein.
      Previous studies have shown that deletion of Isd11 in yeast or silencing of ISD11 in mammalian cells leads to impairment of Fe-S cluster biogenesis and reduction in Fe-S cluster containing enzyme activity (
      • Adam A.C.
      • Bornhövd C.
      • Prokisch H.
      • Neupert W.
      • Hell K.
      The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria.
      ,
      • Shi Y.
      • Ghosh M.C.
      • Tong W.H.
      • Rouault T.A.
      Human ISD11 is essential for both iron-sulfur cluster assembly and maintenance of normal cellular iron homeostasis.
      ). Isd11/ISD11 is an essential protein but its actual role in Fe-S cluster biogenesis by interacting with the sulfur donor Nfs1 is not clear. Importantly, the amino acid residues are critical for Isd11/ISD11 function and are not well characterized. Our genetic analysis maps 8 prominent residues throughout the protein critical for ISD11 function. Intriguingly, one residue (Phe-40) belongs to the predicted N-terminal helix 1 region, whereas the remaining 7 were from helix 3 and the C-terminal region (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76, Leu-81, and Glu-84), thus highlighting important segments of ISD11 for its in vivo function. Based on the severity of growth phenotype and associated biochemical defects, the ISD11 mutants demonstrate a linear correlation of ISD11 function with its ability to form a stable subcomplex with NFS1, thus firmly establishing the indispensable nature of their interaction for a functional Fe-S cluster biogenesis. Interestingly, these observations indicate that ISD11 stabilizes NFS1, predominantly through hydrophobic interaction, considering the nature of the critical amino acid residues. Most of the essential residues mapped in ISD11, which are required for a stable interaction with NFS1 are highly conserved across phylogeny, thereby indicating the critical nature of these interacting residues across genera.
      Based on our results, it is reasonable to believe that ISD11 stabilizes NFS1 through physical interaction, probably shielding the exposed hydrophobic surface. So, the mutual co-dependence of these two proteins might be essential for the initial rate of scaffolding reaction on Isu1/ISCU during Fe-S cluster biogenesis. In line with this argument, in yeast it has been reported that, an inadequate amount of Isd11 results in the steady-state reduction of mitochondrial levels of Nfs1 (
      • Adam A.C.
      • Bornhövd C.
      • Prokisch H.
      • Neupert W.
      • Hell K.
      The Nfs1 interacting protein Isd11 has an essential role in Fe/S cluster biogenesis in mitochondria.
      ,
      • Wiedemann N.
      • Urzica E.
      • Guiard B.
      • Müller H.
      • Lohaus C.
      • Meyer H.E.
      • Ryan M.T.
      • Meisinger C.
      • Mühlenhoff U.
      • Lill R.
      • Pfanner N.
      Essential role of Isd11 in mitochondrial iron-sulfur cluster synthesis on Isu scaffold proteins.
      ). A similar result was obtained for the ts ISD11 mutants as well as in the COXPD19 disease variant (R68A), where Nfs1 levels were reduced as compared with the WT. A plausible reason for the decrease in the Nfs1 levels could be attributed to its relatively lower stability due to the compromised ISD11 interaction, causing aggregation and degradation by proteases in the mitochondrial compartment. Such regulatory mechanisms have been demonstrated in yeast Isu1 mutants, which are defective in the Nfs1 interaction and undergo degradation by Pim1 protease, therefore, modulating the protein levels (
      • Song J.Y.
      • Marszalek J.
      • Craig E.A.
      Cysteine desulfurase Nfs1 and Pim1 protease control levels of Isu, the Fe-S cluster biogenesis scaffold.
      ). In addition to reduced Nfs1 expression, diminished levels of Isu1 was also observed in mutant strains, especially with a stronger ts phenotype and R68A disease variant, implying the paramount nature of these residues for maintaining Isu1 levels as well. However, the steady-state level of the J-protein Jac1, which is involved in the cluster transfer process, was unaltered in mutants, suggesting that impairment of ISD11 function affects the early steps of Fe-S cluster assembly not the late transfer process.
      Sulfur donation is an indispensable and rate-limiting early event during the Fe-S assembly process (
      • Lill R.
      Function and biogenesis of iron-sulphur proteins.
      ). Because ISD11 assists the function of sulfur transfer protein NFS1 by preventing its aggregation, it is reasonable to predict that loss of ISD11 function will influence the rate of Fe-S cluster assembly reaction by impeding the sulfur transfer process. Consistent with this argument, compromised function of ISD11 mutants demonstrates decreased activity of the Fe-S cluster containing enzymes and reduction in their expression levels. An inefficient Fe-S cluster assembly process overall deregulates the maturation of the Fe-S cluster containing proteins, including crucial ETC multienzyme complexes. As a consequence, defective ETC enzymes lead to reduce pumping of protons across the mitochondrial inner membrane, thus decreasing the effective proton motive force required for synthesis of ATP inside mitochondria. Reinforcing the idea, the ISD11 ts mutants and COXPD19-associated disease variant (R68A/R68L) showed reduced mitochondrial membrane potential and diminished respiratory activity with lower ATP levels in the mitochondria. Based on these findings, it is reasonable to believe that reduced respiratory activity associated with the R68L ISD11 mutation in COXPD19 patients may be responsible for the manifestation of disease symptoms, especially the most common symptom of respiratory distress (
      • Lim S.C.
      • Friemel M.
      • Marum J.E.
      • Tucker E.J.
      • Bruno D.L.
      • Riley L.G.
      • Christodoulou J.
      • Kirk E.P.
      • Boneh A.
      • DeGennaro C.M.
      • Springer M.
      • Mootha V.K.
      • Rouault T.A.
      • Leimkühler S.
      • Thorburn D.R.
      • Compton A.G.
      Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes.
      ).
      The inerrant mitochondrial Fe-S cluster biogenesis process is an effective mode of maintenance of cellular iron homeostasis and its toxicity (
      • Richardson D.R.
      • Lane D.J.
      • Becker E.M.
      • Huang M.L.
      • Whitnall M.
      • Suryo Rahmanto Y.
      • Sheftel A.D.
      • Ponka P.
      Mitochondrial iron trafficking and the integration of iron metabolism between the mitochondrion and cytosol.
      ). Defects in this process lead to the elevated mitochondrial iron load. For instance, in case of the neurodegenerative disorder Friedreich's ataxia or exercise intolerance disorder ISCU myopathy where the rate of Fe-S clusters biogenesis process is impaired, there is an increased iron level in the mitochondria (
      • Saha P.P.
      • Kumar S.K.
      • Srivastava S.
      • Sinha D.
      • Pareek G.
      • D'Silva P.
      The presence of multiple cellular defects associated with a novel G50E iron-sulfur cluster scaffold protein (ISCU) mutation leads to development of mitochondrial myopathy.
      ,
      • Babcock M.
      • de Silva D.
      • Oaks R.
      • Davis-Kaplan S.
      • Jiralerspong S.
      • Montermini L.
      • Pandolfo M.
      • Kaplan J.
      Regulation of mitochondrial iron accumulation by Yfh1p, a putative homolog of frataxin.
      ,
      • Cavadini P.
      • Gellera C.
      • Patel P.I.
      • Isaya G.
      Human frataxin maintains mitochondrial iron homeostasis in Saccharomyces cerevisiae.
      ). In agreement with this view, our results highlight that the reduction in Fe-S cluster synthesis in the R68A/R68L mutant leads to accumulation of iron in yeast and human mitochondria. Notably, we also observed a significant enhancement of ROS levels in the R68A/R68L mutant mitochondria in both model systems. This is probably due to the loss of ETC complex activity, causing leakage of free electrons during electron transport and producing superoxide radicals (
      • Batandier C.
      • Fontaine E.
      • Kériel C.
      • Leverve X.M.
      Determination of mitochondrial reactive oxygen species: methodological aspects.
      ,
      • Kudin A.P.
      • Bimpong-Buta N.Y.
      • Vielhaber S.
      • Elger C.E.
      • Kunz W.S.
      Characterization of superoxide-producing sites in isolated brain mitochondria.
      ,
      • Liu Y.
      • Fiskum G.
      • Schubert D.
      Generation of reactive oxygen species by the mitochondrial electron transport chain.
      ). Additionally, enrichment of the reduced form of iron in the mitochondria leads to production of superoxide radicals and highly toxic hydroxyl radicals through Fenton's reaction (
      • Levi S.
      • Rovida E.
      The role of iron in mitochondrial function.
      ,
      • Toyokuni S.
      Iron and carcinogenesis: from Fenton reaction to target genes.
      ). These superoxide and hydroxyl radicals are detrimental to the cellular components as they inflict further damage to the pre-existing Fe-S clusters present in the ETC and other enzyme complexes. This initiates a vicious cycle of ROS production causing oxidative stress, which has been implicated in several pathological conditions (
      • Kirkinezos I.G.
      • Moraes C.T.
      Reactive oxygen species and mitochondrial diseases.
      ,
      • de Moura M.B.
      • dos Santos L.S.
      • Van Houten B.
      Mitochondrial dysfunction in neurodegenerative diseases and cancer.
      ). Moreover, acute oxidative stress also damages the mitochondrial inner membrane potential leading to membrane depolarization, thereby contributing toward reduced respiration and compromised mitochondrial quality control. Based on our experimental findings from both the yeast and mammalian systems, we believe that free radical-mediated oxidative damage plays an additive effect toward the manifestation of complex disease symptoms in COXPD19 patients.
      In conclusion, this report indicates that ISD11 plays a significant role to stabilize NFS1. However, E. coli does not have an ortholog of Isd11 for IscS function, probably attributing to its better intrinsic stability. On the other hand, the reduced intrinsic stability associated with eukaryotic Nfs1/NFS1 may demand an additional interacting partner such as Isd11/ISD11 to attain maximum stability through physical association. This ensures that the steady-state level is maintained in intricate physiological conditions. It will be interesting to have the structure of ISD11 bound to NFS1, as it will be helpful in co-relating the functional aspects to structural details of the protein. This might allow further elucidation of the macromolecular defects associated with the progression of COXPD19 disease as a result of R68L mutation.

      Author Contributions

      P. P. S., P. D. S., and P. K. S. K. designed the study and analyzed the data. P. P. S. performed the experiments and prepared all the figures. S. S. and D. S. performed and analyzed the experiment shown in Fig. 7, E and F. P. P. S., P. D. S., S. S., and D. S wrote the manuscript. All authors reviewed the results and approved the final version of manuscript.

      Acknowledgments

      We thank Dr. Nicholas Pfanner for providing the Δisd11 yeast strain and antibody against aconitase and Rieske Fe-S prteins. We thank Dr. Roland Lill and Dr. Wing-Hang Tong for anti-yeast Nfs1 and anti-human NFS1 antibodies, respectively. We also thank the Flow Cytometry Facility of the Indian Institute of Science, Bangalore, for FACS experiments and the Solid State and Structural Chemistry Unit (SSCU) of the Indian Institute of Science, Bangalore, for AAS analysis.

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