Mitochondrial NADH kinase, Pos5p, is required for efficient iron-sulfur cluster biogenesis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the mitochondrial inner membrane readily allows transport of cytosolic NAD(+), but not NADPH, to the matrix. Pos5p is the only known NADH kinase in the mitochondrial matrix. The enzyme phosphorylates NADH to NADPH and is the major source of NADPH in the matrix. The importance of mitochondrial NADPH for cellular physiology is underscored by the phenotypes of the Δpos5 mutant, characterized by oxidative stress sensitivity and iron-sulfur (Fe-S) cluster deficiency. Fe-S clusters are essential cofactors of proteins such as aconitase [4Fe-4S] and ferredoxin [2Fe-2S] in mitochondria. Intact mitochondria isolated from wild-type yeast can synthesize these clusters and insert them into the corresponding apoproteins. Here, we show that this process of Fe-S cluster biogenesis in wild-type mitochondria is greatly stimulated and kinetically favored by the addition of NAD(+) or NADH in a dose-dependent manner, probably via transport into mitochondria and subsequent conversion into NADPH. Unlike wild-type mitochondria, Δpos5 mitochondria cannot efficiently synthesize Fe-S clusters on endogenous aconitase or imported ferredoxin, although cluster biogenesis in isolated Δpos5 mitochondria is restored to a significant extent by a small amount of imported Pos5p. Interestingly, Fe-S cluster biogenesis in wild-type mitochondria is further enhanced by overexpression of Pos5p. The effects of Pos5p on Fe-S cluster generation in mitochondria indicate that one or more steps in the biosynthetic process require NADPH. The role of mitochondrial NADPH in Fe-S cluster biogenesis appears to be distinct from its function in anti-oxidant defense.     

Adrenodoxin reductase homolog (Arh1p) of yeast mitochondria required for iron homeostasis

Arh1p is an essential mitochondrial protein of yeast with reductase activity. Here we show that this protein is involved in iron metabolism. A yeast strain was constructed in which the open reading frame was placed under the control of a galactose-regulated promoter. Protein expression was induced by galactose and repressed to undetectable levels in the absence of galactose, although cells grew quite well in the absence of inducer. Under noninducing conditions, cellular iron uptake was dysregulated, exhibiting a failure to repress in response to medium iron. Iron trafficking within the cell was also disturbed. Exposure of Arh1p-depleted cells to increasing iron concentrations during growth led to drastic increases in mitochondrial iron, indicating a loss of homeostatic control. Activity of aconitase, a prototype Fe-S protein, was deficient at all concentrations of mitochondrial iron, although the protein level was unaltered. Heme protein deficiencies were exacerbated in the iron-loaded mitochondria, suggesting a toxic side effect of accumulated iron. Finally, a time course correlated the cellular depletion of Arh1p with the coordinated appearance of various mutant phenotypes including dysregulated cellular iron uptake, deficiency of Fe-S protein activities in mitochondria and cytoplasm, and deficiency of hemoproteins. Thus, Arh1p is required for control of cellular and mitochondrial iron levels and for the activities of Fe-S cluster proteins.     

Loss of Cardiolipin Leads to Perturbation of Mitochondrial and Cellular Iron Homeostasis

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.     

The iron-sulphur protein Ind1 is required for effective complex I assembly

NADH:ubiquinone oxidoreductase (complex I) of the mitochondrial inner membrane is a multi-subunit protein complex containing eight iron-sulphur (Fe-S) clusters. Little is known about the assembly of complex I and its Fe-S clusters. Here, we report the identification of a mitochondrial protein with a nucleotide-binding domain, named Ind1, that is required specifically for the effective assembly of complex I. Deletion of the IND1 open reading frame in the yeast Yarrowia lipolytica carrying an internal alternative NADH dehydrogenase resulted in slower growth and strongly decreased complex I activity, whereas the activities of other mitochondrial Fe-S enzymes, including aconitase and succinate dehydrogenase, were not affected. Two-dimensional gel electrophoresis, in vitro activity tests and electron paramagnetic resonance signals of Fe-S clusters showed that only a minor fraction (approximately 20%) of complex I was assembled in the ind1 deletion mutant. Using in vivo and in vitro approaches, we found that Ind1 can bind a [4Fe-4S] cluster that was readily transferred to an acceptor Fe-S protein. Our data suggest that Ind1 facilitates the assembly of Fe-S cofactors and subunits of complex I.     

A role for IOP1 in mammalian cytosolic iron-sulfur protein biogenesis

The biogenesis of cytosolic iron-sulfur (Fe-S) proteins in mammalian cells is poorly understood. In Saccharomyces cerevisiae, there is a pathway dedicated to cytosolic Fe-S protein maturation that involves several essential proteins. One of these is Nar1, which intriguingly is homologous to iron-only hydrogenases, ancient enzymes that catalyze the formation of hydrogen gas in anaerobic bacteria. There are two orthologues of Nar1 in mammalian cells, iron-only hydrogenase-like protein 1 (IOP1) and IOP2 (also known as nuclear prelamin A recognition factor). We examined IOP1 for a potential role in mammalian cytosolic Fe-S protein biogenesis. We found that knockdown of IOP1 in both HeLa and Hep3B cells decreases the activity of cytosolic aconitase, an Fe-S protein, but not that of mitochondrial aconitase. Knockdown of IOP2, in contrast, had no effect on either. The decrease in aconitase activity upon IOP1 knockdown is rescued by expression of a small interference RNA-resistant version of IOP1. Upon loss of its Fe-S cluster, cytosolic aconitase is known to be converted to iron regulatory protein 1, and consistent with this, we found that IOP1 knockdown increases transferrin receptor 1 mRNA levels and decreases ferritin heavy chain protein levels. IOP1 knockdown also leads to a decrease in activity of xanthine oxidase, a distinct cytosolic Fe-S protein. Taken together, these results provide evidence that IOP1 is involved in mammalian cytosolic Fe-S protein maturation.     

Glutathione reductase and a mitochondrial thioredoxin play overlapping roles in maintaining iron-sulfur enzymes in fission yeast

In the fission yeast Schizosaccharomyces pombe, the pgr1+ gene encoding glutathione (GSH) reductase (GR) is essentially required for cell survival. Depletion of GR caused proliferation arrest at the G1 phase of the cell cycle under aerobic conditions. Multicopy suppressors that restore growth were screened, and one effective suppressor was found to be the trx2+ gene, encoding a mitochondrial thioredoxin. This suggests that GR is critically required for some mitochondrial function(s). We found that GR resides in both cytosolic and organellar fractions of the cell. Depletion of GR lowered the respiration rate and the activity of oxidation-labile Fe-S enzymes such as mitochondrial aconitase and cytosolic sulfite reductase. Trx2 did not reverse the high ratio of oxidized glutathione to GSH or the low respiration rate observed in GR-depleted cells. However, it brought the activity of oxidation-labile Fe-S enzymes to a normal level, suggesting that the maintenance of Fe-S enzymes is a critical factor in the survival of S. pombe. The activity of succinate dehydrogenase, an oxidation-insensitive Fe-S enzyme, however, was not affected by GR depletion, suggesting that GR is not required for the biogenesis of the Fe-S cluster. The total iron content was greatly increased by GR depletion and was brought to a nearly normal level by Trx2. These results indicate that the essentiality of GR in the aerobic growth of S. pombe is derived from its role in maintaining oxidation-labile Fe-S enzymes and iron homeostasis.     

Recycling of RNA binding iron regulatory protein 1 into an aconitase after nitric oxide removal depends on mitochondrial ATP

Iron regulatory proteins (IRPs) control iron metabolism by specifically interacting with iron-responsive elements (IREs) on mRNAs. Nitric oxide (NO) converts IRP-1 from a [4Fe-4S] aconitase to a trans-regulatory protein through Fe-S cluster disassembly. Here, we have focused on the fate of IRE binding IRP1 from murine macrophages when NO flux stops. We show that virtually all IRP-1 molecules from NO-producing cells dissociated from IRE and recovered aconitase activity after re-assembling a [4Fe-4S] cluster in vitro. The reverse change in IRP-1 activities also occurred in intact cells no longer exposed to NO and did not require de novo protein synthesis. Likewise, inhibition of mitochondrial aconitase via NO-induced Fe-S cluster disassembly was also reversed independently of protein translation after NO removal. Our results provide the first evidence of Fe-S cluster repair of NO-modified aconitases in mammalian cells. Moreover, we show that reverse change in IRP-1 activities and repair of mitochondrial aconitase activity depended on energized mitochondria. Finally, we demonstrate that IRP-1 activation by NO was accompanied by both a drastic decrease in ferritin levels and an increase in transferrin receptor mRNA levels. However, although ferritin expression was recovered upon IRP-1-IRE dissociation, expression of transferrin receptor mRNA continued to rise for several hours after stopping NO flux.     

Structural investigations by extended X-ray absorption fine structure spectroscopy of the iron center of mitochondrial aconitase in higher plant cells.

We have obtained iron K-edge extended x-ray absorption fine structure spectra of the plant mitochondrial aconitase in its active state, in the presence (aconitase (+)) and absence (aconitase (-)) of the substrate citrate. Analysis of the data indicates that oxygens are present in the first coordination shell, at an average Fe-O distance of 1.96/1.98 A (aconitase (+)/aconitase(-)). Part of these oxygens is provided by the citrate, which binds at 1.99 A from the iron in aconitase (+). The second shell (sulfur) contribution is split and is consistent with Fe-S distances of 2.30/2.29 and 2.56/2.59 A, and the third shell (iron) is consistent with an Fe-Fe distance of 2.83/2.84 A. Both Fe-S and Fe-Fe distances are longer than similar distances found in most Fe-S centers. A strong scattering at approximately 5 A has been identified as originating from an iron atom which is near to, but not part of, the Fe-S cluster. These data indicate that active plant mitochondrial aconitase contains a novel type of iron center.     

Up-regulation of plasma membrane-associated redox activities in neuronal cells lacking functional mitochondria

Mitochondria-deficient cells (rho(o) cells) survive through enhanced glycolytic metabolism in the presence of pyruvate and uridine. The plasma membrane redox system (PMRS) contains several NAD(P)H-related enzymes and plays a key role in maintaining the levels of NAD(+)/NADH and reduced coenzyme Q. In this study, rho(o) cells were used to investigate how the PMRS is regulated under conditions of mitochondrial dysfunction. rho(o) cells exhibited a lower oxygen consumption rate and higher levels of lactate than parental cells, and were more sensitive to glycolysis inhibitors (2-deoxyglucose and iodoacetamide) than control cells. However, they were more resistant to H(2)O(2), consistent with increased catalase activity and decreased oxidative damage (protein carbonyls and nitrotyrosine). PM-associated redox enzyme activities were enhanced in rho(o) cells compared to those in control cells. Our data suggest that all PMRS enzymes and biomarkers tested are closely related to the ability of the PMs to maintain redox homeostasis. These results illustrate that an up-regulated PM redox activity can protect cells from oxidative stress as a result of an improved antioxidant capacity, and suggest a mechanism by which neurons adapt to conditions of impaired mitochondrial function.     

Mitochondrial complex I,aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.     

Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN^?, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]₄ cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]₄ cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]₄ cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.     

真核细胞顺乌头酸酶活性检测新技术——胶内酶活性分析法

顺乌头酸酶（aconitase,Aco）是细胞内重要的铁硫蛋白酶,它催化细胞内柠檬酸经中间产物顺乌头酸生成异柠檬酸. 真核细胞中顺乌头酸酶有两种,分别定位在细胞质的顺乌头酸酶1（c-Aco）和定位在线粒体的顺乌头酸酶2（m-Aco）.检测它们活性的变化能敏感地反映出细胞中能量代谢、自由基产生、铁硫簇组装及铁代谢水平的改变. 顺乌头酸酶活性的传统检测方法通常是测定细胞中总的顺乌头酸酶活性,该方法难以准确区分出c-Aco和m-Aco各自的活性变化.因此我们建立一种胶内酶活性分析法检测顺乌头酸酶活性. 该方法利用非变性电泳技术将c-Aco和m-Aco浓缩分离,通过泡染底物显色,条带颜色深浅反映了酶活性的强弱. 同时,比较了胶内酶活性分析法和分光光度法检测细胞内c-Aco和m-Aco的活性,并对比检测了过氧化氢处理细胞前后Aco活性的变化.结果显示,这两种方法均可敏感地检测出Aco的活性改变,并有广泛的细胞系实用性,但胶内酶活性分析法可区别测定c-Aco和m-Aco活性,不需繁琐的细胞质和线粒体分离,简便易行.文中介绍的线粒体分离纯化技术也为线粒体功能深入研究提供了一个快速、高效的分离纯化方法.     

Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.     

Inactivation of mitochondrial aspartate aminotransferase contributes to the respiratory deficit of yeast frataxin-deficient cells

Friedreich's ataxia is a hereditary neurodegenerative disease caused by reduced expression of mitochondrial frataxin. Frataxin deficiency causes impairment in respiratory capacity, disruption of iron homoeostasis and hypersensitivity to oxidants. Although the redox properties of NAD (NAD+ and NADH) are essential for energy metabolism, only few results are available concerning homoeostasis of these nucleotides in frataxin-deficient cells. In the present study, we show that the malate-aspartate NADH shuttle is impaired in Saccharomyces cerevisiae frataxin-deficient cells (Δyfh1) due to decreased activity of cytosolic and mitochondrial isoforms of malate dehydrogenase and to complete inactivation of the mitochondrial aspartate aminotransferase (Aat1). A considerable decrease in the amount of mitochondrial acetylated proteins was observed in the Δyfh1 mutant compared with wild-type. Aat1 is acetylated in wild-type mitochondria and deacetylated in Δyfh1 mitochondria suggesting that inactivation could be due to this post-translational modification. Mutants deficient in iron-sulfur cluster assembly or lacking mitochondrial DNA also showed decreased activity of Aat1, suggesting that Aat1 inactivation was a secondary phenotype in Δyfh1 cells. Interestingly, deletion of the AAT1 gene in a wild-type strain caused respiratory deficiency and disruption of iron homoeostasis without any sensitivity to oxidative stress. Our results show that secondary inactivation of Aat1 contributes to the amplification of the respiratory defect observed in Δyfh1 cells. Further implication of mitochondrial protein deacetylation in the physiology of frataxin-deficient cells is anticipated.     

Sites and mechanisms of aconitase inactivation by peroxynitrite: modulation by citrate and glutathione

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.