Both human ferredoxins 1 and 2 and ferredoxin reductase are important for iron-sulfur cluster biogenesis

Abstract

Ferredoxins are iron-sulfur proteins that have been studied for decades because of their role in facilitating the monooxygenase reactions catalyzed by p450 enzymes. More recently, studies in bacteria and yeast have demonstrated important roles for ferredoxin and ferredoxin reductase in iron-sulfur cluster assembly. The human genome contains two homologous ferredoxins, ferredoxin 1 (FDX1) and ferredoxin 2 (FDX2--formerly known as ferredoxin 1L). More recently, the roles of these two human ferredoxins in iron-sulfur cluster assembly were assessed, and it was concluded that FDX1 was important solely for its interaction with p450 enzymes to synthesize mitochondrial steroid precursors, whereas FDX2 was used for synthesis of iron-sulfur clusters, but not steroidogenesis. To further assess the role of the FDX-FDXR system in mammalian iron-sulfur cluster biogenesis, we performed siRNA studies on FDX1 and FDX2, on several human cell lines, using oligonucleotides identical to those previously used, along with new oligonucleotides that specifically targeted each gene. We concluded that both FDX1 and FDX2 were important in iron-sulfur cluster biogenesis. Loss of FDX1 activity disrupted activity of iron-sulfur cluster enzymes and cellular iron homeostasis, causing mitochondrial iron overload and cytosolic iron depletion. Moreover, knockdown of the sole human ferredoxin reductase, FDXR, diminished iron-sulfur cluster assembly and caused mitochondrial iron overload in conjunction with cytosolic depletion. Our studies suggest that interference with any of the three related genes, FDX1, FDX2 or FDXR, disrupts iron-sulfur cluster assembly and maintenance of normal cytosolic and mitochondrial iron homeostasis.

Fig. 1

Silencing of either FDX1 or its homologue, FDX2, diminishes aconitase activities and activates iron-responsive element (IRE)-binding protein activities. (A) Protein sequence alignment of human FDX1 and FDX2. Identical residues are marked byasterisks, and similar residues are denoted by dots. (B) Comparison of mRNA change to evaluate specificity in FDX1 or FDX2 knock-downs in HeLa cells. (C) In-gel aconitase assays revealed activity of mitochondrial and cytosolic aconitases in HeLa cells treated with FDX1 and FDX2 siRNA (WT, wild-type; (–)ctrl, negative control; oligo, siRNA of either FDX1 or FDX2) after two successive transfections. (D) Gel retardation assays of IRPs. Transfected cells were harvested six days after two successive transfections and analyzed for total binding activity of IRP1 and IRP2 to ³²P-labeled IRE of human ferritin mRNA. Lysates of WT, FDX1 and FDX2 knock-down HeLa cells (10 mg protein/lane) were incubated with ³²P-IRE and resolved on a 10% non-denaturing gel. Lanes were: WT, wild type; (–) ctrl, negative control; oligo, siRNAs of FDX1 or FDX2 as labeled. IRP1 is the bottom band and IRP2 is the upper band of the IRP-IRE complexes. Western blots for IRP1 and α-tubulin (loading control) demonstrate equal loading of the gels.

Fig. 2

Depletion of FDX1 results in iron accumulation within mitochondria. After fractionation, the iron contents were determined (described in Materials and methods), and are displayed in the unit of μg/dl [Fe²⁺]. Data represent the mean ± SE (n=3). Analysis of variance (ANOVA) was used for the statistical analysis, with P-value <0.05 considered to be significant. The results shown are representative of data from three independent experiments that displayed similar results. Statistical analyses of the data from these three experiments showed that there was no significant difference between wild-type and negative control, since the mitochondrial iron contents were 4.9 ± 0.02 and 4.8 ± 0.03 under the normal medium culture condition, whereas, mitochondrial iron contents were 5.52 ± 0.01 and 5.48 ± 0.02 when cells were grown in FAC-supplemented medium. However, the mitochondrial iron contents were 11.04 ± 0.02 for oligo1-treated cells, 9.08 ± 0.03 for oligo2-treated cells under the conditions of normal medium culture, and with FAC supplementation, the mitochondrial iron contents were 89.17 ± 0.49 for oligo1-treated cells and 78.15±0.62 for oligo2-treated cells. These values are significantly different (P<0.05) from those of RNAi oligo control-treated cells, as calculated by ANOVA followed by two-tail Dunett's test.

Fig. 3

FDX1 knock-down leads to increased IRP2, and indications of cytosolic iron depletion. (A) FDX1 knock-downs were performed using four independent oligos and both mitochondria and cytololic aconitase activities were diminished after three or four successive transfections. Aconitase activity assay revealed reduced activity of mitochondrial and cytosolic aconitases in HeLa cells treated with FDX1 siRNA (Lanes are WT, wild-type; (–)ctrl, negative control represent cells treated with Lipofectamine 2000 reagent; oligos 1–4 represent different siRNAs of FDX1). (B) Comparison of protein expression levels between FDX1 knock-down HeLa cells and wild-type and negative control HeLa cells. At the protein expression level, western blots of cells transfected with newly designed FDX1 siRNA revealed that IRP1 and α-tubulin levels (loading control) did not significantly change, IRP2 and TfR1 protein levels increased, whereas ferritin levels decreased significantly. Results shown are representative of four independent experiments. (C) mRNA levels of IRP2, TfR1 (transferrin receptor 1) IRP1, and FPN. (D) Xanthine oxidase activity assay in the RNAi-treated cells compared with the untreated wild-type and negative control cells. (E) Lactate dehydrogenase activity assay in the RNAi-treated cells compared with the untreated wild-type and negative control cells.

Fig. 4

FDX1 depletion diminished complex I activity and induced expression of the mitochondrial superoxide dismutase. (A) Amounts of complexes I, II and III and SOD2 were assessed after FDX1 depletion. (B) Complex I enzymatic activity decreased in HeLa cells after the FDX1 depletion. Results represent the average of 3 repeats, and differences were statistically significant (p<0.05).

Fig. 5

FDX1 is essential for heme biosynthesis in the human erythoblast cell line, K562. (A) Both FDX1 protein and mRNA levels were decreased upon FDX1 depletion in K562 cells. (B) Compared with wild-type and negative control K562 cells, FDX1 knock-down led to increased IRP2, TfR1 and FPN protein expression, whereas FTN protein expression levels decreased. (C) At mRNA levels, FDX1 knock-down led to increased expression of TfR1 mRNA, but there was no change of FTN mRNA. MFRN mRNA levels increased. (D) Decreased heme content was detected in FDX1-depleted K562 cells compared with wild-type and negative control K562 cells. (E) Upon FDX1 depletion, FECH and HMOX1 protein levels increased, whereas ALAS2 protein levels decreased. (F) Upon FDX1 depletion, FECH mRNA level increased.

Fig. 6

siRNA treatments of the sole human ferredoxin reductase, FDXR, cause decreased iron–sulfur protein activities and activation of IRPs. Using oligonucleotides, we decreased FDXR protein (6A) and mRNA levels (6B, left). After three successive transfections, activities of m-aco and c-aco were reduced. This experiment is representative of three experimental repeats. IRE binding activities of IRP1 and IRP2 increased on gel-shift assay (6D) and xanthine oxidase activity decreased significantly (6E), whereas lactate dehydrogenase activity did not change (6F).

Fig. 7

Knockdown of FDXR results in mitochondrial iron overload in HeLa cells. Using oligos 1 or 2, the iron content of mitochondria increased in HeLa cells grown in normal media, and more markedly in iron-supplemented media.

Fig. 8

FDXR knockdown resulted in cytosolic iron depletion and increased expression of the mitochondrial iron importer, MFRN1. On western blots, we observed increased IRP2 and TfR1, decreased ferritin (FTN), and increased mitoferrin (MFRN1) (8A). On western blot, we also noted decreased NTHL, an iron–sulfur cluster containing endonuclease and decreased cytochrome C, a mitochondrial heme protein (8B). Complex I activity was significantly decreased in FDXR knockdowns (8C).

Fig. 9

Heme was reduced in FDXR knockdown in HCT116 (K562) cells. Heme levels were significantly reduced (9A), by knockdown of FDXR protein (9B) and mRNA levels (9C), but ferrochelatase mRNA levels were increased (9C). In western blots, IRP2 levels increased, TfR1 increased, but ALAS2 protein levels decreased (9D).