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• W2070374460 abstract "The human mitochondrial outer membrane protein mitoNEET is a novel target of the type II diabetes drug pioglitazone. The C-terminal cytosolic domain of mitoNEET hosts a redox-active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine residues and one histidine residue. Here we report that human mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells. In vitro studies show that purified mitoNEET [2Fe-2S] clusters can be partially reduced by monothiols such as reduced glutathione, l-cysteine or N-acetyl-l-cysteine and fully reduced by dithiothreitol or the E. coli thioredoxin/thioredoxin reductase system under anaerobic conditions. Importantly, thiol-reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by hydrogen peroxide without disruption of the clusters in vitro and in E. coli cells, indicating that mitoNEET may act as a sensor of oxidative signals to regulate mitochondrial functions via its [2Fe-2S] clusters. Furthermore, the binding of the type II diabetes drug pioglitazone in mitoNEET effectively inhibits the thiol-mediated reduction of [2Fe-2S] clusters, suggesting that pioglitazone may modulate the function of mitoNEET by blocking the thiol-mediated reduction of [2Fe-2S] clusters in the protein. The human mitochondrial outer membrane protein mitoNEET is a novel target of the type II diabetes drug pioglitazone. The C-terminal cytosolic domain of mitoNEET hosts a redox-active [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine residues and one histidine residue. Here we report that human mitoNEET [2Fe-2S] clusters are fully reduced when expressed in Escherichia coli cells. In vitro studies show that purified mitoNEET [2Fe-2S] clusters can be partially reduced by monothiols such as reduced glutathione, l-cysteine or N-acetyl-l-cysteine and fully reduced by dithiothreitol or the E. coli thioredoxin/thioredoxin reductase system under anaerobic conditions. Importantly, thiol-reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by hydrogen peroxide without disruption of the clusters in vitro and in E. coli cells, indicating that mitoNEET may act as a sensor of oxidative signals to regulate mitochondrial functions via its [2Fe-2S] clusters. Furthermore, the binding of the type II diabetes drug pioglitazone in mitoNEET effectively inhibits the thiol-mediated reduction of [2Fe-2S] clusters, suggesting that pioglitazone may modulate the function of mitoNEET by blocking the thiol-mediated reduction of [2Fe-2S] clusters in the protein. The human mitochondrial protein mitoNEET is a novel target of the type II diabetes drugs thiazolidinediones, such as pioglitazone (1.Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Identification of a novel mitochondrial protein (“mitoNEET”) cross-linked specifically by a thiazolidinedione photoprobe.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E252-E260Crossref PubMed Scopus (270) Google Scholar). Genetic studies have shown that the deletion of mitoNEET in mice results in a reduced oxidative phosphorylation capacity in mitochondria (2.Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar). Although an increased expression of mitoNEET in adipocytes in mice enhances lipid uptake and storage and inhibits mitochondrial iron transport into the matrix, depletion of mitoNEET in adipocytes leads to less weight gain in mice (3.Kusminski C.M. Holland W.L. Sun K. Park J. Spurgin S.B. Lin Y. Askew G.R. Simcox J.A. McClain D.A. Li C. Scherer P.E. MitoNEET-driven alterations in adipocyte mitochondrial activity reveal a crucial adaptive process that preserves insulin sensitivity in obesity.Nat. Med. 2012; 18: 1539-1549Crossref PubMed Scopus (317) Google Scholar), suggesting that mitoNEET may regulate the energy metabolism in mitochondria (1.Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Identification of a novel mitochondrial protein (“mitoNEET”) cross-linked specifically by a thiazolidinedione photoprobe.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E252-E260Crossref PubMed Scopus (270) Google Scholar). Recent studies further indicated that mitoNEET may have a central role in neurodegenerative diseases (4.Yonutas H.M. Sullivan P.G. Targeting PPAR isoforms following CNS injury.Curr. Drug Targets. 2013; 14: 733-742Crossref PubMed Scopus (29) Google Scholar, 5.Geldenhuys W.J. Van der Schyf C.J. Rationally designed multi-targeted agents against neurodegenerative diseases.Curr. Med. Chem. 2013; 20: 1662-1672Crossref PubMed Scopus (83) Google Scholar) and breast cancer proliferation (6.Sohn Y.S. Tamir S. Song L. Michaeli D. Matouk I. Conlan A.R. Harir Y. Holt S.H. Shulaev V. Paddock M.L. Hochberg A. Cabanchick I.Z. Onuchic J.N. Jennings P.A. Nechushtai R. Mittler R. NAF-1 and mitoNEET are central to human breast cancer proliferation by maintaining mitochondrial homeostasis and promoting tumor growth.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 14676-14681Crossref PubMed Scopus (140) Google Scholar) by maintaining mitochondrial homeostasis in cells. MitoNEET localizes on mitochondrial outer membranes (2.Wiley S.E. Murphy A.N. Ross S.A. van der Geer P. Dixon J.E. MitoNEET is an iron-containing outer mitochondrial membrane protein that regulates oxidative capacity.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 5318-5323Crossref PubMed Scopus (218) Google Scholar) via the N-terminal transmembrane α-helix (residues 14–32) (1.Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Identification of a novel mitochondrial protein (“mitoNEET”) cross-linked specifically by a thiazolidinedione photoprobe.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E252-E260Crossref PubMed Scopus (270) Google Scholar). Expression of the soluble C-terminal domain (residues 33–108) of mitoNEET in Escherichia coli cells produced a protein that contains a [2Fe-2S] cluster (7.Wiley S.E. Paddock M.L. Abresch E.C. Gross L. van der Geer P. Nechushtai R. Murphy A.N. Jennings P.A. Dixon J.E. The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster.J. Biol. Chem. 2007; 282: 23745-23749Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Crystallographic studies revealed that mitoNEET exists as a homodimer, with each monomer hosting a [2Fe-2S] cluster via an unusual ligand arrangement of three cysteine residues (Cys-72, Cys-74, and Cys-83) and one histidine residue (His-87) (8.Hou X. Liu R. Ross S. Smart E.J. Zhu H. Gong W. Crystallographic studies of human MitoNEET.J. Biol. Chem. 2007; 282: 33242-33246Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9.Lin J. Zhou T. Ye K. Wang J. Crystal structure of human mitoNEET reveals distinct groups of iron-sulfur proteins.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 14640-14645Crossref PubMed Scopus (96) Google Scholar, 10.Paddock M.L. Wiley S.E. Axelrod H.L. Cohen A.E. Roy M. Abresch E.C. Capraro D. Murphy A.N. Nechushtai R. Dixon J.E. Jennings P.A. MitoNEET is a uniquely folded 2Fe 2S outer mitochondrial membrane protein stabilized by pioglitazone.Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 14342-14347Crossref PubMed Scopus (211) Google Scholar). The [2Fe-2S] clusters in mitoNEET are redox-active, with a midpoint redox potential at pH 7 (Em7) of ∼0 mV (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar, 12.Tirrell T.F. Paddock M.L. Conlan A.R. Smoll Jr., E.J. Nechushtai R. Jennings P.A. Kim J.E. Resonance Raman studies of the (His)(Cys)(3) 2Fe-2S cluster of MitoNEET. Comparison to the (Cys)(4) mutant and implications of the effects of pH on the labile metal center.Biochemistry. 2009; 48: 4747-4752Crossref PubMed Scopus (42) Google Scholar). Substitution of the unique His-87 with cysteine in mitoNEET shifts the Em7 value of the [2Fe-2S] clusters from 0 to −320 mV (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar) and increases the stability of the cluster in the protein (13.Conlan A.R. Paddock M.L. Homer C. Axelrod H.L. Cohen A.E. Abresch E.C. Zuris J.A. Nechushtai R. Jennings P.A. Mutation of the His ligand in mitoNEET stabilizes the 2Fe-2S cluster despite conformational heterogeneity in the ligand environment.Acta Crystallogr. D. Biol. Crystallogr. 2011; 67: 516-523Crossref PubMed Scopus (24) Google Scholar). The redox property and stability of the [2Fe-2S] clusters in mitoNEET are also modulated by the type II diabetes drug pioglitazone (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar), excess zinc (14.Tan G. Landry A.P. Dai R. Wang L. Lu J. Ding H. Competition of zinc ion for the [2Fe-2S] cluster binding site in the diabetes drug target protein mitoNEET.Biometals. 2012; 25: 1177-1184Crossref PubMed Scopus (11) Google Scholar), resveratrol-3-sulfate (15.Arif W. Xu S. Isailovic D. Geldenhuys W.J. Carroll R.T. Funk M.O. Complexes of the outer mitochondrial membrane protein mitoNEET with resveratrol-3-sulfate.Biochemistry. 2011; 50: 5806-5811Crossref PubMed Scopus (19) Google Scholar), NADPH (16.Zhou T. Lin J. Feng Y. Wang J. Binding of reduced nicotinamide adenine dinucleotide phosphate destabilizes the iron-sulfur clusters of human mitoNEET.Biochemistry. 2010; 49: 9604-9612Crossref PubMed Scopus (26) Google Scholar), the interdomain interactions (17.Baxter E.L. Jennings P.A. Onuchic J.N. Interdomain communication revealed in the diabetes drug target mitoNEET.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 5266-5271Crossref PubMed Scopus (21) Google Scholar, 18.Baxter E.L. Jennings P.A. Onuchic J.N. Strand swapping regulates the iron-sulfur cluster in the diabetes drug target mitoNEET.Proc. Natl. Acad. Sci. U.S.A. 2012; 109: 1955-1960Crossref PubMed Scopus (18) Google Scholar), and the hydrogen bond network in the protein (19.Bak D.W. Elliott S.J. Conserved hydrogen bonding networks of MitoNEET Tune Fe-S cluster binding and structural stability.Biochemistry. 2013; 52: 4687-4696Crossref PubMed Scopus (38) Google Scholar), suggesting that mitoNEET may act as a sensor of multiple signals to regulate mitochondrial functions (19.Bak D.W. Elliott S.J. Conserved hydrogen bonding networks of MitoNEET Tune Fe-S cluster binding and structural stability.Biochemistry. 2013; 52: 4687-4696Crossref PubMed Scopus (38) Google Scholar). Interestingly, purified mitoNEET can also transfer the [2Fe-2S] clusters to apo-protein with a 50% completion time of about 120 min at room temperature (20.Zuris J.A. Harir Y. Conlan A.R. Shvartsman M. Michaeli D. Tamir S. Paddock M.L. Onuchic J.N. Mittler R. Cabantchik Z.I. Jennings P.A. Nechushtai R. Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 13047-13052Crossref PubMed Scopus (102) Google Scholar, 21.Zuris J.A. Ali S.S. Yeh H. Nguyen T.A. Nechushtai R. Paddock M.L. Jennings P.A. NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.J. Biol. Chem. 2012; 287: 11649-11655Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Because mitochondria are the primary sites for iron-sulfur cluster biogenesis (22.Lill R. Function and biogenesis of iron-sulphur proteins.Nature. 2009; 460: 831-838Crossref PubMed Scopus (835) Google Scholar), it is appealing to consider that mitoNEET may participate in the iron-sulfur cluster biogenesis process in human cells (20.Zuris J.A. Harir Y. Conlan A.R. Shvartsman M. Michaeli D. Tamir S. Paddock M.L. Onuchic J.N. Mittler R. Cabantchik Z.I. Jennings P.A. Nechushtai R. Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 13047-13052Crossref PubMed Scopus (102) Google Scholar, 21.Zuris J.A. Ali S.S. Yeh H. Nguyen T.A. Nechushtai R. Paddock M.L. Jennings P.A. NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.J. Biol. Chem. 2012; 287: 11649-11655Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Nevertheless, the iron-sulfur cluster transfer occurs only when the [2Fe-2S] clusters in mitoNEET are oxidized (20.Zuris J.A. Harir Y. Conlan A.R. Shvartsman M. Michaeli D. Tamir S. Paddock M.L. Onuchic J.N. Mittler R. Cabantchik Z.I. Jennings P.A. Nechushtai R. Facile transfer of [2Fe-2S] clusters from the diabetes drug target mitoNEET to an apo-acceptor protein.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 13047-13052Crossref PubMed Scopus (102) Google Scholar) and the cluster transfer process is inhibited by NADPH (21.Zuris J.A. Ali S.S. Yeh H. Nguyen T.A. Nechushtai R. Paddock M.L. Jennings P.A. NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.J. Biol. Chem. 2012; 287: 11649-11655Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), suggesting that the mitoNEET-mediated iron-sulfur cluster transfer could be regulated by the redox state of the [2Fe-2S] clusters and by intracellular NADPH. Because the cytosolic redox potential in eukaryotic cells is estimated to be around −325 mV (pH 7.0) (23.Dooley C.T. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar), it is expected that the mitoNEET [2Fe-2S] clusters (Em7 = 0 mV) (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar, 12.Tirrell T.F. Paddock M.L. Conlan A.R. Smoll Jr., E.J. Nechushtai R. Jennings P.A. Kim J.E. Resonance Raman studies of the (His)(Cys)(3) 2Fe-2S cluster of MitoNEET. Comparison to the (Cys)(4) mutant and implications of the effects of pH on the labile metal center.Biochemistry. 2009; 48: 4747-4752Crossref PubMed Scopus (42) Google Scholar) would be in a reduced state in cells under physiological conditions. Nevertheless, the redox state of the mitoNEET [2Fe-2S] clusters in cells has not been demonstrated, and specific cellular components that may reduce the mitoNEET [2Fe-2S] clusters have not been identified. Here we report that human mitoNEET [2Fe-2S] clusters are in the fully reduced state when expressed in E. coli cells under normal growth conditions and that purified mitoNEET [2Fe-2S] clusters can be partially reduced by monothiols such as reduced glutathione, l-cysteine, and N-acetyl-l-cysteine and fully reduced by dithiothreitol and the E. coli thioredoxin-1 reduced by thioredoxin reductase and NADPH. Importantly, the reduced mitoNEET [2Fe-2S] clusters can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, indicating that the redox state of the mitoNEET [2Fe-2S] clusters can be modulated by biological thiols and oxidative signals. Furthermore, we find that the type II diabetes drug pioglitazone can effectively inhibit the thiol-mediated reduction of the mitoNEET [2Fe-2S] clusters in vitro, suggesting that pioglitazone may modulate the function of mitoNEET by blocking the thiol-mediated reduction of the [2Fe-2S] clusters in the protein. The gene encoding human mitoNEET33–108 (containing amino acid residues 33–108) was cloned previously from the human cDNA library as described in Ref. 14.Tan G. Landry A.P. Dai R. Wang L. Lu J. Ding H. Competition of zinc ion for the [2Fe-2S] cluster binding site in the diabetes drug target protein mitoNEET.Biometals. 2012; 25: 1177-1184Crossref PubMed Scopus (11) Google Scholar. The mitoNEET mutant in which histidine 87 was substituted with cysteine (H87C) was constructed using the QuikChange site-directed mutagenesis kit (Stratagene). The constructed mutation was confirmed by direct sequencing (Operon Co.). Human mitoNEET and the mitoNEET mutant proteins were prepared following procedures described previously (14.Tan G. Landry A.P. Dai R. Wang L. Lu J. Ding H. Competition of zinc ion for the [2Fe-2S] cluster binding site in the diabetes drug target protein mitoNEET.Biometals. 2012; 25: 1177-1184Crossref PubMed Scopus (11) Google Scholar). E. coli thioredoxin-1 (TrxA) and thioredoxin reductase (TrxB) were produced from E. coli cells using the expression vectors pDL59 (24.Veine D.M. Mulrooney S.B. Wang P.F. Williams Jr., C.H. Formation and properties of mixed disulfides between thioredoxin reductase from Escherichia coli and thioredoxin. Evidence that cysteine-138 functions to initiate dithiol-disulfide interchange and to accept the reducing equivalent from reduced flavin.Protein Sci. 1998; 7: 1441-1450Crossref PubMed Scopus (32) Google Scholar) and pTrR301 (25.Mulrooney S.B. Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation.Protein Expr. Purif. 1997; 9: 372-378Crossref PubMed Scopus (40) Google Scholar), respectively, and purified as described in Ref. 26.Ding H. Harrison K. Lu J. Thioredoxin reductase system mediates iron binding in IscA and iron delivery for the iron-sulfur cluster assembly in IscU.J. Biol. Chem. 2005; 280: 30432-30437Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar. Both E. coli thioredoxin and thioredoxin reductase were purified as the native form. The purity of purified proteins was greater than 95%, as judged by electrophoresis analysis on a 15% polyacrylamide gel containing SDS, followed by staining with Coomassie Blue. The protein concentration of purified mitoNEET or the mitoNEET mutant H87C was measured at 280 nm using an extinction coefficient of 8.6 mm−1cm−1. The protein concentration of thioredoxin-1 and thioredoxin reductase was determined at 280 nm using extinction coefficients of 14.2 and 17.7 mm−1cm−1, respectively. A Beckman DU640 UV-visible spectrometer equipped with a temperature control was used for measuring absorption spectra. Pioglitazone, l-cysteine, N-acetyl-l-cysteine, reduced glutathione, and other chemicals were purchased from Sigma. Isopropyl β-d-1-thiogalactopyranoside, NADPH, kanamycin, ampicillin, and dithiothreitol were from Research Product International Co. Pioglitazone was dissolved in dimethyl sulfoxide as a stock solution containing 25 mm pioglitazone. An equal amount of dimethyl sulfoxide was added to the samples as a control. For whole-cell EPR measurements, E. coli cells were grown for 2 h before the recombinant protein expression was induced. After 2 h of protein expression, cells were harvested, washed once with phosphate buffer (pH 7.5), and resuspended in phosphate buffer or fresh LB medium. An aliquot (350 μl) of the E. coli cells was then transferred to the EPR tube and frozen immediately in liquid nitrogen. For the reduced samples, freshly prepared sodium dithionite was added to the E. coli cells before the EPR samples were prepared. For protein sample preparation, purified mitoNEET [2Fe-2S] clusters dissolved in buffer containing 20 mm Tris (pH 8.0) and 500 mm NaCl were purged with pure argon gas in sealed vials for 15 min before reductant was added under anaerobic conditions. After incubation at 37 °C for 20 min, samples were transferred to the EPR tubes and frozen immediately in liquid nitrogen. The X-band EPR spectra were recorded using a Bruker model ESR-300 spectrometer equipped with an Oxford Instruments 910 continuous flow cryostat. Routine EPR conditions were as follows: microwave frequency, 9.47 GHz; microwave power, 10.0 milliwatt; modulation frequency, 100 kHz; modulation amplitude, 1.2 millitesla; temperature, 20 K; receive gain, 105. Purified human mitoNEET [2Fe-2S] clusters are in an oxidized state and have no EPR signals (Fig. 1A). When purified mitoNEET [2Fe-2S]] clusters are reduced with sodium dithionite (Em7 = -600 mV (27.Mayhew S.G. The redox potential of dithionite and SO-2 from equilibrium reactions with flavodoxins, methyl viologen and hydrogen plus hydrogenase.Eur. J. Biochem. 1978; 85: 535-547Crossref PubMed Scopus (416) Google Scholar)), a rhombic EPR spectrum with gx= 1.895, gy=1.937, and gz = 2.005 appears (Fig. 1A), as reported previously (7.Wiley S.E. Paddock M.L. Abresch E.C. Gross L. van der Geer P. Nechushtai R. Murphy A.N. Jennings P.A. Dixon J.E. The outer mitochondrial membrane protein mitoNEET contains a novel redox-active 2Fe-2S cluster.J. Biol. Chem. 2007; 282: 23745-23749Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 19.Bak D.W. Elliott S.J. Conserved hydrogen bonding networks of MitoNEET Tune Fe-S cluster binding and structural stability.Biochemistry. 2013; 52: 4687-4696Crossref PubMed Scopus (38) Google Scholar, 28.Dicus M.M. Conlan A. Nechushtai R. Jennings P.A. Paddock M.L. Britt R.D. Stoll S. Binding of histidine in the (Cys)(3)(His)(1)-coordinated [2Fe-2S] cluster of human mitoNEET.J. Am. Chem. Soc. 2010; 132: 2037-2049Crossref PubMed Scopus (59) Google Scholar, 29.Iwasaki T. Samoilova R.I. Kounosu A. Ohmori D. Dikanov S.A. Continuous-wave and pulsed EPR characterization of the [2Fe-2S](Cys)3(His)1 cluster in rat MitoNEET.J. Am. Chem. Soc. 2009; 131: 13659-13667Crossref PubMed Scopus (26) Google Scholar). The gx, gy, and gz values represent the anisotropic g-factors of a typical [2Fe-2S] cluster in proteins. In parallel, we also prepared the mitoNEET mutant H87C, in which the unique ligand His-87 of the [2Fe-2S] cluster is replaced with Cys. Although purified mitoNEET H87C has no EPR signal, addition of sodium dithionite produces a new rhombic EPR spectrum of the [2Fe-2S] cluster, with gx = 1.896, gy = 1.970, and gz = 1.992, that is distinct from that of the wild-type mitoNEET (Fig. 1A). The EPR measurement provides an efficient and non-intrusive approach to determine the redox state of iron-sulfur clusters in proteins in solutions or in whole cells (30.Ding H. Demple B. In vivo kinetics of a redox-regulated transcriptional switch.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 8445-8449Crossref PubMed Scopus (116) Google Scholar, 31.Djaman O. Outten F.W. Imlay J.A. Repair of oxidized iron-sulfur clusters in Escherichia coli.J. Biol. Chem. 2004; 279: 44590-44599Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Here, we took the advantage of EPR to explore the redox state of the mitoNEET [2Fe-2S] clusters expressed in E. coli cells. Fig. 1B shows that E. coli cells without any recombinant proteins have very little or no EPR signals because of low concentrations of endogenous iron-sulfur proteins in cells (30.Ding H. Demple B. In vivo kinetics of a redox-regulated transcriptional switch.Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 8445-8449Crossref PubMed Scopus (116) Google Scholar, 31.Djaman O. Outten F.W. Imlay J.A. Repair of oxidized iron-sulfur clusters in Escherichia coli.J. Biol. Chem. 2004; 279: 44590-44599Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Under the same experimental conditions, E. coli cells expressing human mitoNEET show a rhombic EPR signal at g = 1.94, which is identical to that of the reduced mitoNEET [2Fe-2S] clusters (Fig. 1A). In contrast, E. coli cells expressing the mitoNEET mutant H87C do not have any EPR signals (Fig. 1B), although both mitoNEET and the mitoNEET mutant H87C are expressed similarly in E. coli cells on the basis of SDS-PAGE gel analysis (data not shown). This is likely because the mutation of His-87 to Cys shifts the Em7 value of the mitoNEET [2Fe-2S] clusters from 0 to −320 mV (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar). Because the cytosolic redox potential of E. coli cells is about −260 mV (32.Gaudu P. Weiss B. SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 10094-10098Crossref PubMed Scopus (184) Google Scholar), the mitoNEET mutant H87C [2Fe-2S] clusters would be mostly in an oxidized (EPR-silent) state in E. coli cells. To further examine the redox state of the [2Fe-2S] clusters in wild-type mitoNEET and the mitoNEET mutant H87C expressed in E. coli cells, excess sodium dithionite is added to the cells to reduce all iron-sulfur proteins. Fig. 1C shows that addition of sodium dithionite to E. coli cells without recombinant proteins does not produce any new EPR signals. On the other hand, addition of sodium dithionite to E. coli cells expressing the mitoNEET mutant H87C generates an EPR signal at g = 1.97, which is the same as that of the reduced mitoNEET mutant H87C [2Fe-2S] clusters (Fig. 1A). Thus, the oxidized [2Fe-2S] clusters in the mitoNEET mutant H87C in E. coli cells can be reduced by sodium dithionite. In contrast, the EPR signal at g = 1.94 of the E. coli cells expressing the wild-type mitoNEET is not changed by addition of sodium dithionite, suggesting that the wild-type mitoNEET [2Fe-2S] clusters are fully reduced in E. coli cells even before the addition of sodium dithionite. Because the cytosolic redox potential in eukaryotic cells (-325 mV (23.Dooley C.T. Dore T.M. Hanson G.T. Jackson W.C. Remington S.J. Tsien R.Y. Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.J. Biol. Chem. 2004; 279: 22284-22293Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar)) is about 65 mV lower than that in E. coli cells (-260 mV (32.Gaudu P. Weiss B. SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 10094-10098Crossref PubMed Scopus (184) Google Scholar)), we postulate that the mitoNEET [2Fe-2S] clusters are most likely in a reduced state in human cells under physiological conditions. Although it has been shown that the mitoNEET [2Fe-2S] clusters are redox-active (11.Bak D.W. Zuris J.A. Paddock M.L. Jennings P.A. Elliott S.J. Redox characterization of the FeS protein MitoNEET and impact of thiazolidinedione drug binding.Biochemistry. 2009; 48: 10193-10195Crossref PubMed Scopus (62) Google Scholar, 12.Tirrell T.F. Paddock M.L. Conlan A.R. Smoll Jr., E.J. Nechushtai R. Jennings P.A. Kim J.E. Resonance Raman studies of the (His)(Cys)(3) 2Fe-2S cluster of MitoNEET. Comparison to the (Cys)(4) mutant and implications of the effects of pH on the labile metal center.Biochemistry. 2009; 48: 4747-4752Crossref PubMed Scopus (42) Google Scholar), specific biological molecules that can reduce or oxidize the mitoNEET [2Fe-2S] clusters are unknown. Because the mitoNEET [2Fe-2S] clusters are fully reduced in E. coli cells, we reason that the mitoNEET [2Fe-2S] clusters may be reduced by cellular reducing components that are common in both human and E. coli cells. However, NADPH is not one of them because NADPH fails to reduce the mitoNEET [2Fe-2S] clusters in vitro (21.Zuris J.A. Ali S.S. Yeh H. Nguyen T.A. Nechushtai R. Paddock M.L. Jennings P.A. NADPH inhibits [2Fe-2S] cluster protein transfer from diabetes drug target MitoNEET to an apo-acceptor protein.J. Biol. Chem. 2012; 287: 11649-11655Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Among other cellular reducing components, biological thiols are abundant. More importantly, biological thiols are able to form relatively stable intermediate thiol free radicals (33.Quijano C. Alvarez B. Gatti R.M. Augusto O. Radi R. Pathways of peroxynitrite oxidation of thiol groups.Biochem. J. 1997; 322: 167-173Crossref PubMed Scopus (232) Google Scholar, 34.Eaton P. Protein thiol oxidation in health and disease. Techniques for measuring disulfides and related modifications in complex protein mixtures.Free Radic. Biol. Med. 2006; 40: 1889-1899Crossref PubMed Scopus (231) Google Scholar) that can potentially provide electrons for the single-electron reduction of the oxidized [2Fe-2S] clusters in mitoNEET. To test this idea, purified mitoNEET was incubated with thiols under anaerobic conditions. After incubation, the samples were subjected to EPR and UV-visible absorption measurements. The reduced mitoNEET [2Fe-2S] clusters were observed from the EPR signal at g = 1.94, and the UV-visible absorption peaks at 420 and 550 nm. As shown in Fig. 2, all biological thiols tested (reduced glutathione, l-cysteine, and N-acetyl-l-cysteine) are able to partially reduce the mitoNEET [2Fe-2S] clusters. Reduced glutathione appears to have the least ability to reduce the mitoNEET [2Fe-2S] clusters. l-cysteine and N-acetyl-l-cysteine are able to reduce about 50% of the mitoNEET [2Fe-2S] clusters in solution under anaerobic conditions, in comparison with the fully reduced mitoNEET [2Fe-2S] clusters by sodium dithionite. A further increase of l-cysteine concentration (up to 50-fold excess over the mitoNEET [2Fe-2S] clusters) or incubation time (up to 60 min) does not increase the amount of the reduced mitoNEET [2Fe-2S] clusters in the incubation solution (data not shown), suggesting that l-cysteine could not fully reduce the mitoNEET [2Fe-2S] clusters under the experimental conditions. Interestingly, incubation wi" @default.
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• W2070374460 title "Redox Control of Human Mitochondrial Outer Membrane Protein MitoNEET [2Fe-2S] Clusters by Biological Thiols and Hydrogen Peroxide" @default.
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