Iron-sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in Escherichiacoli.

It was generally postulated that when intracellular free iron content is elevated in bacteria, the ferric uptake regulator (Fur) binds its corepressor a mononuclear ferrous iron to regulate intracellular iron homeostasis. However, the proposed iron-bound Fur had not been identified in any bacteria. In previous studies, we have demonstrated that Escherichia coli Fur binds a [2Fe-2S] cluster in response to elevation of intracellular free iron content and that binding of the [2Fe-2S] cluster turns on Fur as an active repressor to bind a specific DNA sequence known as the Fur-box. Here we find that the iron-sulfur cluster assembly scaffold protein IscU is required for the [2Fe-2S] cluster assembly in Fur, as deletion of IscU inhibits the [2Fe-2S] cluster assembly in Fur and prevents activation of Fur as a repressor in E. coli cells in response to elevation of intracellular free iron content. Additional studies reveal that IscU promotes the [2Fe-2S] cluster assembly in apo-form Fur and restores its Fur-box binding activity in vitro. While IscU is also required for the [2Fe-2S] cluster assembly in the Haemophilus influenzae Fur in E. coli cells, deletion of IscU does not significantly affect the [2Fe-2S] cluster assembly in the E. coli ferredoxin and siderophore-reductase FhuF. Our results suggest that IscU may have a unique role for the [2Fe-2S] cluster assembly in Fur and that regulation of intracellular iron homeostasis is closely coupled with iron-sulfur cluster biogenesis in E. coli.

The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli.

Escherichia coli ferric uptake regulator (Fur) binds a [2Fe-2S] cluster, not a mononuclear iron, when the intracellular free iron content is elevated in E. coli cells. Here we report that the C-terminal domain (residues 83-148) of E. coli Fur (Fur-CTD) is sufficient to bind the [2Fe-2S] cluster in response to elevation of the intracellular free iron content in E. coli cells. Deletion of gene fur in E. coli cells increases the intracellular free iron content and promotes the [2Fe-2S] cluster binding in the Fur-CTD in the cells grown in LB medium under aerobic growth conditions. When the Fur-CTD is expressed in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, the Fur-CTD also progressively binds a [2Fe-2S] cluster with a maximum occupancy of about 36%. Like the E. coli Fur-CTD, the CTD of the Haemophilus influenzae Fur can also bind a [2Fe-2S] cluster in wild type E. coli cells grown in M9 medium supplemented with increasing concentrations of iron, indicating that binding of the [2Fe-2S] cluster in the C-terminal domain is highly conserved among Fur proteins. The results suggest that the Fur-CTD can be used as a physiological probe to assess the intracellular free iron content in bacteria.

Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli.

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). It has been postulated that when intracellular free iron content is elevated, Fur binds ferrous iron to downregulate the genes for iron uptake. However, the iron-bound Fur had not been identified in any bacteria until we recently found that Escherichia coli Fur binds a [2Fe-2S] cluster, but not a mononuclear iron, in E. coli mutant cells that hyperaccumulate intracellular free iron. Here, we report that E. coli Fur also binds a [2Fe-2S] cluster in wildtype E. coli cells grown in M9 medium supplemented with increasing concentrations of iron under aerobic growth conditions. Additionally, we find that binding of the [2Fe-2S] cluster in Fur turns on its binding activity for specific DNA sequences known as the Fur-box and that removal of the [2Fe-2S] cluster from Fur eliminates its Fur-box binding activity. Mutation of the conserved cysteine residues Cys-93 and Cys-96 to Ala in Fur results in the Fur mutants that fail to bind the [2Fe-2S] cluster, have a diminished binding activity for the Fur-box in vitro, and are inactive to complement the function of Fur in vivo. Our results suggest that Fur binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular free iron content in E. coli cells.

Nitric oxide reversibly binds the reduced [2Fe-2S] cluster in mitochondrial outer membrane protein mitoNEET and inhibits its electron transfer activity.

MitoNEET is a mitochondrial outer membrane protein that regulates energy metabolism, iron homeostasis, and production of reactive oxygen species in cells. Aberrant expression of mitoNEET in tissues has been linked to type II diabetes, neurodegenerative diseases, and several types of cancer. Structurally, the N-terminal domain of mitoNEET has a single transmembrane alpha helix that anchors the protein to mitochondrial outer membrane. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine and one histidine residues. Here we report that the reduced [2Fe-2S] cluster in the C-terminal cytosolic domain of mitoNEET (mitoNEET45-108) is able to bind nitric oxide (NO) without disruption of the cluster. Importantly, binding of NO at the reduced [2Fe-2S] cluster effectively inhibits the redox transition of the cluster in mitoNEET45-108. While the NO-bound [2Fe-2S] cluster in mitoNEET45-108 is stable, light excitation releases NO from the NO-bound [2Fe-2S] cluster and restores the redox transition activity of the cluster in mitoNEET45-108. The results suggest that NO may regulate the electron transfer activity of mitoNEET in mitochondrial outer membrane via reversible binding to its reduced [2Fe-2S] cluster.

Electron transfer activity of the nanodisc-bound mitochondrial outer membrane protein mitoNEET.

MitoNEET is the first iron-sulfur protein found in mitochondrial outer membrane. Abnormal expression of mitoNEET in cells has been linked to several types of cancer, type II diabetes, and neurodegenerative diseases. Structurally, mitoNEET is anchored to mitochondrial outer membrane via its N-terminal single transmembrane alpha helix. The C-terminal cytosolic domain of mitoNEET binds a [2Fe-2S] cluster via three cysteine and one histidine residues. It has been shown that mitoNEET has a crucial role in energy metabolism, iron homeostasis, and free radical production in cells. However, the exact function of mitoNEET remains elusive. Previously, we reported that the C-terminal soluble domain of mitoNEET has a specific binding site for flavin mononucleotide (FMN) and can transfer electrons from FMNH2 to oxygen or ubiquinone-2 via its [2Fe-2S] cluster. Here we have constructed a hybrid protein using the N-terminal transmembrane domain of Escherichia coli YneM and the C-terminal soluble domain of human mitoNEET and assembled the hybrid protein YneM-mitoNEET into phospholipid nanodiscs. The results show that the [2Fe-S] clusters in the nanodisc-bound YneM-mitoNEET can be rapidly reduced by FMNH2 which is reduced by flavin reductase using NADH as the electron donor. Addition of lumichrome, a FMN analog, effectively inhibits the FMNH2-mediated reduction of the [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET. The reduced [2Fe-2S] clusters in the nanodisc-bound YneM-mitoNEET are quickly oxidized by oxygen under aerobic conditions or by ubiquinone-10 in the nanodiscs under anaerobic conditions. Because NADH oxidation is required for cellular glycolytic activity, we propose that the mitochondrial outer membrane protein mitoNEET may promote glycolysis by transferring electrons from FMNH2 to oxygen or ubiquinone-10 in mitochondria.

Ferric uptake regulators (Fur) from Vibrio cholerae and Helicobacter pylori bind a [2Fe-2S] cluster in response to elevation of intracellular free iron content.

Intracellular iron homeostasis in bacteria is primarily regulated by ferric uptake regulator (Fur). Since its discovery, Fur has been assumed to bind ferrous iron and regulate expression of target genes. However, the iron-bound Fur has never been isolated from any bacteria. In previous studies, we have shown that Escherichia coli Fur and Haemophilus influenzae Fur bind a [2Fe-2S] cluster via the conserved Cys-93 and Cys-96 when expressed in the E. coli mutant cells in which intracellular free iron content is elevated. Here we report that Fur homologs from Vibrio cholerae and Helicobacter pylori which contain Cys-93 and Cys-96 can also bind a [2Fe-2S] cluster. On the other hand, Fur homolog from Magnetospirillum gryphiswaldense MSR-1 which has no cysteine residues fails to bind any [2Fe-2S] clusters. Interestingly, different Fur proteins with the conserved Cys-93 and Cys-96 have distinct binding activities for the [2Fe-2S] cluster, with H. influenzae Fur having the highest, followed by E. coli Fur, V. cholera Fur, and H. pylori Fur. Binding of the [2Fe-2S] cluster in the Fur proteins is significantly decreased when expressed in wild-type E. coli cells, indicating that binding of the [2Fe-2S] clusters in Fur proteins is regulated by the levels of intracellular free iron content. Finally, unlike the [2Fe-2S] clusters in E. coli ferredoxin, the [2Fe-2S] clusters in the Fur proteins are not stable and quickly release ferrous iron when the clusters are reduced, suggesting that Fur may undergo reversible binding of the [2Fe-2S] cluster in response to intracellular free iron content in bacteria.

Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli.

The ferric uptake regulator (Fur) is a global transcription factor that regulates intracellular iron homeostasis in bacteria. The current hypothesis states that when the intracellular "free" iron concentration is elevated, Fur binds ferrous iron, and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins. However, the "iron-bound" Fur has never been isolated from any bacteria. Here we report that the Escherichia coli Fur has a bright red color when expressed in E. coli mutant cells containing an elevated intracellular free iron content because of deletion of the iron-sulfur cluster assembly proteins IscA and SufA. The acid-labile iron and sulfide content analyses in conjunction with the EPR and Mössbauer spectroscopy measurements and the site-directed mutagenesis studies show that the red Fur protein binds a [2Fe-2S] cluster via conserved cysteine residues. The occupancy of the [2Fe-2S] cluster in Fur protein is ∼31% in the E. coli iscA/sufA mutant cells and is decreased to ∼4% in WT E. coli cells. Depletion of the intracellular free iron content using the membrane-permeable iron chelator 2,2´-dipyridyl effectively removes the [2Fe-2S] cluster from Fur in E. coli cells, suggesting that Fur senses the intracellular free iron content via reversible binding of a [2Fe-2S] cluster. The binding of the [2Fe-2S] cluster in Fur appears to be highly conserved, because the Fur homolog from Hemophilus influenzae expressed in E. coli cells also reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis.

Exploring the FMN binding site in the mitochondrial outer membrane protein mitoNEET.

MitoNEET is a mitochondrial outer membrane protein that hosts a redox active [2Fe-2S] cluster in the C-terminal cytosolic domain. Increasing evidence has shown that mitoNEET has an essential role in regulating energy metabolism in human cells. Previously, we reported that the [2Fe-2S] clusters in mitoNEET can be reduced by the reduced flavin mononucleotide (FMNH2) and oxidized by oxygen or ubiquinone-2, suggesting that mitoNEET may act as a novel redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone. Here, we explore the FMN binding site in mitoNEET by using FMN analogs and find that lumiflavin, like FMN, at nanomolar concentrations can mediate the redox transition of the mitoNEET [2Fe-2S] clusters in the presence of flavin reductase and NADH (100 µM) under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that both FMN and lumiflavin can dramatically change the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters and form a covalently bound complex with mitoNEET under blue light exposure, suggesting that FMN/lumiflavin has specific interactions with the [2Fe-2S] clusters in mitoNEET. In contrast, lumichrome, another FMN analog, fails to mediate the redox transition of the mitoNEET [2Fe-2S] clusters and has no effect on the EPR spectrum of the reduced mitoNEET [2Fe-2S] clusters under blue light exposure. Instead, lumichrome can effectively inhibit the FMNH2-mediated reduction of the mitoNEET [2Fe-2S] clusters, indicating that lumichrome may act as a potential inhibitor to block the electron transfer activity of mitoNEET.

Light-induced release of nitric oxide from the nitric oxide-bound CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2.

Human mitochondrial matrix protein Miner2 hosts two [2Fe-2S] clusters via two CDGSH (Cys-Asp-Gly-Ser-His) motifs. Unlike other iron-sulfur clusters in proteins, the reduced CDGSH-type [2Fe-2S] clusters in Miner2 are able to bind nitric oxide (NO) and form stable NO-bound [2Fe-2S] clusters without disruption of the clusters. Here we report that the NO-bound Miner2 [2Fe-2S] clusters can quickly release NO upon the visible light excitation. The UV-visible and Electron Paramagnetic Resonance (EPR) measurements show that the NO-bound Miner2 [2Fe-2S] clusters are converted to the reduced Miner2 [2Fe-2S] clusters upon the light excitation under anaerobic conditions, suggesting that NO binding in the reduced Miner2 [2Fe-2S] clusters is reversible. Additional studies reveal that binding of NO effectively inhibits the redox transition of the Miner2 [2Fe-2S] clusters, indicating that NO may modulate the physiological activity of Miner2 in mitochondria by directly binding to the CDGSH-type [2Fe-2S] clusters in the protein.

Zinc Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

While zinc is an essential trace metal in biology, excess zinc is toxic to organisms. Previous studies have shown that zinc toxicity is associated with disruption of the [4Fe-4S] clusters in various dehydratases in Escherichia coli Here, we report that the intracellular zinc overload in E. coli cells inhibits iron-sulfur cluster biogenesis without affecting the preassembled iron-sulfur clusters in proteins. Among the housekeeping iron-sulfur cluster assembly proteins encoded by the gene cluster iscSUA-hscBA-fdx-iscX in E. coli cells, the scaffold IscU, the iron chaperone IscA, and ferredoxin have strong zinc binding activity in cells, suggesting that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by binding to the iron-sulfur cluster assembly proteins. Mutations of the conserved cysteine residues to serine in IscA, IscU, or ferredoxin completely abolish the zinc binding activity of the proteins, indicating that zinc can compete with iron or iron-sulfur cluster binding in IscA, IscU, and ferredoxin and block iron-sulfur cluster biogenesis. Furthermore, intracellular zinc overload appears to emulate the slow-growth phenotype of the E. coli mutant cells with deletion of the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin. Our results suggest that intracellular zinc overload inhibits iron-sulfur cluster biogenesis by targeting the iron-sulfur cluster assembly proteins IscU, IscA, and ferredoxin in E. coli cells.IMPORTANCE Zinc toxicity has been implicated in causing various human diseases. High concentrations of zinc can also inhibit bacterial cell growth. However, the underlying mechanism has not been fully understood. Here, we report that zinc overload in Escherichia coli cells inhibits iron-sulfur cluster biogenesis by targeting specific iron-sulfur cluster assembly proteins. Because iron-sulfur proteins are involved in diverse physiological processes, the zinc-mediated inhibition of iron-sulfur cluster biogenesis could be largely responsible for the zinc-mediated cytotoxicity. Our finding provides new insights on how intracellular zinc overload may inhibit cellular functions in bacteria.

Electron transfer kinetics of the mitochondrial outer membrane protein mitoNEET.

Increasing evidence suggests that the mitochondrial outer membrane protein mitoNEET is a key regulator of energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Previously, we reported that mitoNEET is a redox enzyme that catalyzes electron transfer from the reduced flavin mononucleotide (FMNH2) to oxygen or ubiquinone via its unique [2Fe-2S] clusters. Here, we explore the reduction and oxidation kinetics of the mitoNEET [2Fe-2S] clusters under anaerobic and aerobic conditions. We find that the mitoNEET [2Fe-2S] clusters are rapidly reduced by a catalytic amount of FMNH2 which is reduced by flavin reductase and an equivalent amount of NADH under anaerobic conditions. When the reduced mitoNEET [2Fe-2S] clusters are exposed to air, the [2Fe-2S] clusters are slowly oxidized by oxygen at a rate constant of about 6.0 M-1 s-1. Compared with oxygen, ubiquinone-2 has a much higher activity to oxidize the reduced mitoNEET [2Fe-2S] clusters at a rate constant of about 3.0 × 103 M-1 s-1 under anaerobic conditions. Under aerobic conditions, the mitoNEET [2Fe-2S] clusters can still be reduced by FMNH2 in the presence of flavin reductase and excess NADH. However, when NADH is completely consumed, the reduced mitoNEET [2Fe-2S] clusters are gradually oxidized by oxygen. Addition of ubiquinone-2 also rapidly oxidizes the pre-reduced mitoNEET [2Fe-2S] clusters and effectively prevents the FMNH2-mediated reduction of the mitoNEET [2Fe-2S] clusters under aerobic conditions. The results suggest that ubiquinone may act as an intrinsic oxidant of the reduced mitoNEET [2Fe-2S] clusters in mitochondria under aerobic and anaerobic conditions.

Anaerobic Copper Toxicity and Iron-Sulfur Cluster Biogenesis in Escherichia coli.

While copper is an essential trace element in biology, pollution of groundwater from copper has become a threat to all living organisms. Cellular mechanisms underlying copper toxicity, however, are still not fully understood. Previous studies have shown that iron-sulfur proteins are among the primary targets of copper toxicity in Escherichia coli under aerobic conditions. Here, we report that, under anaerobic conditions, iron-sulfur proteins in E. coli cells are even more susceptible to copper in medium. Whereas addition of 0.2 mM copper(II) chloride to LB (Luria-Bertani) medium has very little or no effect on iron-sulfur proteins in wild-type E. coli cells under aerobic conditions, the same copper treatment largely inactivates iron-sulfur proteins by blocking iron-sulfur cluster biogenesis in the cells under anaerobic conditions. Importantly, proteins that do not have iron-sulfur clusters (e.g., fumarase C and cysteine desulfurase) in E. coli cells are not significantly affected by copper treatment under aerobic or anaerobic conditions, indicating that copper may specifically target iron-sulfur proteins in cells. Additional studies revealed that E. coli cells accumulate more intracellular copper under anaerobic conditions than under aerobic conditions and that the elevated copper content binds to the iron-sulfur cluster assembly proteins IscU and IscA, which effectively inhibits iron-sulfur cluster biogenesis. The results suggest that the copper-mediated inhibition of iron-sulfur proteins does not require oxygen and that iron-sulfur cluster biogenesis is the primary target of anaerobic copper toxicity in cells.IMPORTANCE Copper contamination in groundwater has become a threat to all living organisms. However, cellular mechanisms underlying copper toxicity have not been fully understood up to now. The work described here reveals that iron-sulfur proteins in Escherichia coli cells are much more susceptible to copper in medium under anaerobic conditions than they are under aerobic conditions. Under anaerobic conditions, E. coli cells accumulate excess intracellular copper, which specifically targets iron-sulfur proteins by blocking iron-sulfur cluster biogenesis. Since iron-sulfur proteins are involved in diverse and vital physiological processes, inhibition of iron-sulfur cluster biogenesis by copper disrupts multiple cellular functions and ultimately inhibits cell growth. The results from this study illustrate a new interplay between intracellular copper toxicity and iron-sulfur cluster biogenesis in bacterial cells under anaerobic conditions.

The mitochondrial outer membrane protein mitoNEET is a redox enzyme catalyzing electron transfer from FMNH2 to oxygen or ubiquinone.

Increasing evidence suggests that mitoNEET, a target of the type II diabetes drug pioglitazone, is a key regulator of energy metabolism in mitochondria. MitoNEET is anchored to the mitochondrial outer membrane via its N-terminal α helix domain and hosts a redox-active [2Fe-2S] cluster in its C-terminal cytosolic region. The mechanism by which mitoNEET regulates energy metabolism in mitochondria, however, is not fully understood. Previous studies have shown that mitoNEET specifically interacts with the reduced flavin mononucleotide (FMNH2) and that FMNH2 can quickly reduce the mitoNEET [2Fe-2S] clusters. Here we report that the reduced mitoNEET [2Fe-2S] clusters can be readily oxidized by oxygen. In the presence of FMN, NADH, and flavin reductase, which reduces FMN to FMNH2 using NADH as the electron donor, mitoNEET mediates oxidation of NADH with a concomitant reduction of oxygen. Ubiquinone-2, an analog of ubiquinone-10, can also oxidize the reduced mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. Compared with oxygen, ubiquinone-2 is more efficient in oxidizing the mitoNEET [2Fe-2S] clusters, suggesting that ubiquinone could be an intrinsic electron acceptor of the reduced mitoNEET [2Fe-2S] clusters in mitochondria. Pioglitazone or its analog NL-1 appears to inhibit the electron transfer activity of mitoNEET by forming a unique complex with mitoNEET and FMNH2 The results suggest that mitoNEET is a redox enzyme that may promote oxidation of NADH to facilitate enhanced glycolysis in the cytosol and that pioglitazone may regulate energy metabolism in mitochondria by inhibiting the electron transfer activity of mitoNEET.

Binding of Nitric Oxide in CDGSH-type [2Fe-2S] Clusters of the Human Mitochondrial Protein Miner2.

Iron-sulfur proteins are among the primary targets of nitric oxide in cells. Previous studies have shown that iron-sulfur clusters hosted by cysteine residues in proteins are readily disrupted by nitric oxide forming a protein-bound dinitrosyl iron complex, thiolate-bridged di-iron tetranitrosyl complex, or octanitrosyl cluster. Here we report that human mitochondrial protein Miner2 [2Fe-2S] clusters can bind nitric oxide without disruption of the clusters. Miner2 is a member of a new CDGSH iron-sulfur protein family that also includes two mitochondrial proteins: the type II diabetes-related mitoNEET and the Wolfram syndrome 2-linked Miner1. Miner2 contains two CDGSH motifs, and each CDGSH motif hosts a [2Fe-2S] cluster via three cysteine and one histidine residues. Binding of nitric oxide in the reduced Miner2 [2Fe-2S] clusters produces a major absorption peak at 422 nm without releasing iron or sulfide from the clusters. The EPR measurements and mass spectrometry analyses further reveal that nitric oxide binds to the reduced [2Fe-2S] clusters in Miner2, with each cluster binding one nitric oxide. Although the [2Fe-2S] cluster in purified human mitoNEET and Miner1 fails to bind nitric oxide, a single mutation of Asp-96 to Val in mitoNEET or Asp-123 to Val in Miner1 facilitates nitric oxide binding in the [2Fe-2S] cluster, indicating that a subtle change of protein structure may switch mitoNEET and Miner1 to bind nitric oxide. The results suggest that binding of nitric oxide in the CDGSH-type [2Fe-2S] clusters in mitochondrial protein Miner2 may represent a new nitric oxide signaling mode in cells.

Flavin nucleotides act as electron shuttles mediating reduction of the [2Fe-2S] clusters in mitochondrial outer membrane protein mitoNEET.

MitoNEET, a primary target of type II diabetes drug pioglitazone, has an essential role in regulating energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Structurally, mitoNEET is anchored to the mitochondrial outer membrane via its N-terminal transmembrane α-helix. The C-terminal cytosolic domain of mitoNEET hosts a redox active [2Fe-2S] cluster via three cysteine and one histidine residues. Here we report that the reduced flavin nucleotides can rapidly reduce the mitoNEET [2Fe-2S] clusters under anaerobic or aerobic conditions. In the presence of NADH and flavin reductase, 1 molecule of flavin nucleotide is sufficient to reduce about 100 molecules of the mitoNEET [2Fe-2S] clusters in 4min under aerobic conditions. The electron paramagnetic resonance (EPR) measurements show that flavin mononucleotide (FMN), but not flavin adenine dinucleotide (FAD), has a specific interaction with mitoNEET. Molecular docking models further reveal that flavin mononucleotide binds mitoNEET at the region between the N-terminal transmembrane α-helix and the [2Fe-2S] cluster binding domain. The closest distance between the [2Fe-2S] cluster and the bound flavin mononucleotide in mitoNEET is about 10Å, which could facilitate rapid electron transfer from the reduced flavin nucleotide to the [2Fe-2S] cluster in mitoNEET. The results suggest that flavin nucleotides may act as electron shuttles to reduce the mitoNEET [2Fe-2S] clusters and regulate mitochondrial functions in human cells.

Deletion of the Proposed Iron Chaperones IscA/SufA Results in Accumulation of a Red Intermediate Cysteine Desulfurase IscS in Escherichia coli.

In Escherichia coli, sulfur in iron-sulfur clusters is primarily derived from L-cysteine via the cysteine desulfurase IscS. However, the iron donor for iron-sulfur cluster assembly remains elusive. Previous studies have shown that, among the iron-sulfur cluster assembly proteins in E. coli, IscA has a unique and strong iron-binding activity and that the iron-bound IscA can efficiently provide iron for iron-sulfur cluster assembly in proteins in vitro, indicating that IscA may act as an iron chaperone for iron-sulfur cluster biogenesis. Here we report that deletion of IscA and its paralog SufA in E. coli cells results in the accumulation of a red-colored cysteine desulfurase IscS under aerobic growth conditions. Depletion of intracellular iron using a membrane-permeable iron chelator, 2,2'-dipyridyl, also leads to the accumulation of red IscS in wild-type E. coli cells, suggesting that the deletion of IscA/SufA may be emulated by depletion of intracellular iron. Purified red IscS has an absorption peak at 528 nm in addition to the peak at 395 nm of pyridoxal 5'-phosphate. When red IscS is oxidized by hydrogen peroxide, the peak at 528 nm is shifted to 510 nm, which is similar to that of alanine-quinonoid intermediate in cysteine desulfurases. Indeed, red IscS can also be produced in vitro by incubating wild-type IscS with excess L-alanine and sulfide. The results led us to propose that deletion of IscA/SufA may disrupt the iron delivery for iron-sulfur cluster biogenesis, therefore impeding sulfur delivery by IscS, and result in the accumulation of red IscS in E. coli cells.

Reduction of mitochondrial protein mitoNEET [2Fe-2S] clusters by human glutathione reductase.

The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe-2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe-2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe-2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe-2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe-2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe-2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe-2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe-2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.

The N-terminal domain of human DNA helicase Rtel1 contains a redox active iron-sulfur cluster.

Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed in Escherichia coli cells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of -248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNA in vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

Copper binding in IscA inhibits iron-sulphur cluster assembly in Escherichia coli.

Among the iron-sulphur cluster assembly proteins encoded by gene cluster iscSUA-hscBA-fdx in Escherichia coli, IscA has a unique and strong iron binding activity and can provide iron for iron-sulphur cluster assembly in proteins in vitro. Deletion of IscA and its paralogue SufA results in an E. coli mutant that fails to assemble [4Fe-4S] clusters in proteins under aerobic conditions, suggesting that IscA has a crucial role for iron-sulphur cluster biogenesis. Here we report that among the iron-sulphur cluster assembly proteins, IscA also has a strong and specific binding activity for Cu(I) in vivo and in vitro. The Cu(I) centre in IscA is stable and resistant to oxidation under aerobic conditions. Mutation of the conserved cysteine residues that are essential for the iron binding in IscA abolishes the copper binding activity, indicating that copper and iron may share the same binding site in the protein. Additional studies reveal that copper can compete with iron for the metal binding site in IscA and effectively inhibits the IscA-mediated [4Fe-4S] cluster assembly in E. coli cells. The results suggest that copper may not only attack the [4Fe-4S] clusters in dehydratases, but also block the [4Fe-4S] cluster assembly in proteins by targeting IscA in cells.