The Outer Mitochondrial Membrane Protein mitoNEET Contains a Novel Redox-active 2Fe-2S Cluster

The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252–E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318–5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions. The outer mitochondrial membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252–E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318–5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the outer mitochondrial membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions. Recent studies have revealed a critical role for mitochondrial function in glucose-stimulated insulin secretion (1Maechler P. Carobbio S. Rubi B. Int. J. Biochem. Cell Biol. 2006; 38: 696-709Crossref PubMed Scopus (106) Google Scholar). There is also a growing body of evidence implicating mitochondrial dysfunction in the development of insulin resistance and type 2 diabetes (2Lowell B.B. Shulman G.I. Science. 2005; 307: 384-387Crossref PubMed Scopus (1605) Google Scholar). Diabetic patients demonstrate evidence of oxidative stress (3Mehta J.L. Rasouli N. Sinha A.K. Molavi B. Int. J. Biochem. Cell Biol. 2006; 38: 794-803Crossref PubMed Scopus (145) Google Scholar) and have reduced mitochondrial mass and oxidative capacity in skeletal muscle (4Kelley D.E. He J. Menshikova E.V. Ritov V.B. Diabetes. 2002; 51: 2944-2950Crossref PubMed Scopus (1770) Google Scholar, 5Ritov V.B. Menshikova E.V. He J. Ferrell R.E. Goodpaster B.H. Kelley D.E. Diabetes. 2005; 54: 8-14Crossref PubMed Scopus (676) Google Scholar). Pioglitazone is a member of the thiazolidinedione class of insulin-sensitizing drugs frequently used to treat type 2 diabetes (6Hofmann C.A. Colca J.R. Diabetes Care. 1992; 15: 1075-1078Crossref PubMed Scopus (129) Google Scholar, 7Colca J.R. Biochem. Pharmacol. 2006; 72: 125-131Crossref PubMed Scopus (43) Google Scholar). The thiazolidinedione drugs have traditionally been thought to function as ligands for the peroxisome proliferator-activated receptor γ (8Bogacka I. Xie H. Bray G.A. Smith S.R. Diabetes Care. 2004; 27: 1660-1667Crossref PubMed Scopus (177) Google Scholar, 9Vasudevan A.R. Balasubramanyam A. Diabetes Technol. Ther. 2004; 6: 850-863Crossref PubMed Scopus (90) Google Scholar), although it is unclear whether peroxisome proliferator-activated receptor γ is the sole target of this family of drugs (10Feinstein D.L. Spagnolo A. Akar C. Weinberg G. Murphy P. Gavrilyuk V. Dello Russo C. Biochem. Pharmacol. 2005; 70: 177-188Crossref PubMed Scopus (239) Google Scholar). Using tagged derivatives of pioglitazone incubated with mitochondrial lysates, Colca and colleagues identified a single cross-linked 17-kDa protein (11Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Am. J. Physiol. 2004; 286 (–E260): E252Crossref PubMed Scopus (270) Google Scholar). They named the protein mitoNEET based on its location in the mitochondria and the presence of the amino acid sequence Asn-Glu-Glu-Thr (NEET) near the C terminus (11Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Am. J. Physiol. 2004; 286 (–E260): E252Crossref PubMed Scopus (270) Google Scholar). We have shown that mitoNEET is an integral outer mitochondrial membrane protein (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). It is localized to the mitochondria by an N-terminal targeting sequence, which acts as a membrane tether, resulting in the majority of the protein being exposed to the cytoplasm (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). mitoNEET belongs to a small family of proteins whose hallmark is the presence of a unique CDGSH domain. We refer to the other members of this family as Miner1 and Miner2 (for mitoNEET-related 1 and 2) (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). Both mitoNEET and Miner1 have one CDGSH domain, whereas Miner2 contains two. Although originally annotated as a zinc finger, the CDGSH domain, which has the consensus sequence CXCX2(S/T)X3PXCDG(S/A/T) H, was shown to preferentially bind iron (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). Mitochondria from the hearts of mitoNEET-null mice demonstrated reduced oxidative capacity, suggesting a critical role for mitoNEET in proper mitochondrial function (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). Here we investigated the properties of the recombinant mitoNEET protein. We found that the CDGSH domain of mitoNEET harbors a redox-sensitive 2Fe-2S cluster that is surprisingly pH-labile. Although there are more than 100 families of Fe-S-containing proteins, mitoNEET is unusual in having a Cys-3–His-1 coordination sphere for the 2Fe-2S cluster. Construction of Bacterial Expression Plasmids—Construction of the pET28b-MBP-His-mitoNEET27–108 plasmid, 3The abbreviations used are: MBP, myelin basic protein; EPR, electron paramagnetic resonance; PIPES, 1,4-piperazinediethanesulfonic acid; Tricine, N-2-hydroxy-1,1-bis(hydroxymethyl)ethylglycine; CHES, 2-(cyclohexylamino)ethanesulfonic acid; CAPS, 3-(cyclohexylamino)propanesulfonic acid. encoding amino acids 27–108 of the mitoNEET protein, was previously described (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). The D84N, H87C, and H87Q mutants were generated in the pET28b-MBP-His-mitoNEET27–108 plasmid by site-directed mutagenesis using PCR. Recombinant human mitoNEET33–108 (lacking epitope tags) was generated by PCR and cloned into the pET21a+ vector. The C72S, C74S, and C83S mutants of mitoNEET33–108 were generated by site-directed mutagenesis and cloned into the pET21a+ vector in-frame with the C-terminal His tag. For simplicity, the tagged and untagged proteins are collectively referred to as Δ-mitoNEET. Expression and Purification of Δ-mitoNEET Fusion Proteins in Escherichia coli—Expression of wild-type MBP-His-mitoNEET27–108 and mutants in BL21-CodonPlus-RIL was as described previously (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). The Cys/Ser mutants of Δ-mi-toNEET were grown as above, induced overnight at 23 °C, and (Ni-NTA)-purified using nickel-nitrilotriacetic acid agarose following the manufacturer’s protocol (Qiagen). Growth and expression of untagged mitoNEET33–108 protein was the same as that of MBP-His-mitoNEET27–108, but the induction was extended to 7 h. The bacteria were lysed in Tb buffer (50 mm Tris-HCl, pH 8.0, 0.1% β-mercaptoethanol (v/v)) and clarified by centrifugation. All centrifugations were at 31,000 × g for 20 min. The Δ-mitoNEET protein was purified from the lysate using a series of salt precipitations. After an initial 25% (NH4)2SO4 cut, mitoNEET was pelleted with 75% (NH4)2SO4, resuspended in Tb buffer containing 25% (saturating) (NH4)2SO4 and cleared by centrifugation. Following dialysis against Tb buffer, the Δ-mitoNEET solution was loaded onto an SP-Toyopearl 650M cation exchange column and rinsed with Tb buffer until the eluant was free of protein. The Δ-mitoNEET was eluted with 0.2 m NaCl in Tb buffer. At this point, the eluant had a well defined peak at 458 nm and an optical ratio (A278/A458) of ∼3–4. The sample was dialyzed against TbN buffer (100 mm Tris-HCl, pH 8.0, 0.1% β-mercaptoethanol (v/v), 50 mm NaCl) for storage at 4 °C. The purity of all proteins was evaluated by SDS-PAGE, and concentrations were calculated using the extinction coefficients under denaturing conditions. The integrity of the holo-mitoNEET protein with the cluster was evaluated using UV-VIS spectroscopy. Optical and Electron Paramagnetic Resonance (EPR) Spectroscopy—The optical spectra of recombinant MBP-His-mitoNEET27–108 was measured from the near UV to the near IR (250–1000 nm) on a Cary50 spectrometer (10–20 μm protein in 50 mm Tris, pH 8.0, and 50 mm NaCl). Chemical reduction of Δ-mitoNEET was achieved by adding 2 mm dithionite to the protein solution. Reoxidation of Δ-mitoNEET was achieved by equilibrating with ambient O2 for 1 h. The stability of the Δ-mitoNEET Fe-S cluster was monitored at 458 nm. Its decomposition was measured by an absorbance decrease over time under various pH conditions in a buffer mixture (5 mm each of citrate, PIPES, Tricine, CHES, and CAPS). The spectra for MBP-His alone was featureless above 400 nm (data not shown). EPR spectra of MBP-His-mitoNEET27–108 were measured in both the oxidized and the dithionite-reduced states using ∼100 μm protein in 100 μl of Tb buffer in a Bruker Elexys E500 spectrometer. Following a change in color due to the addition of a few grains of solid dithionite, samples were submerged in liquid nitrogen until analysis on a Bruker EPR spectrometer at 9 GHz and low temperature (15°K). Mass Spectrometry—Untagged mitoNEET33–108 (0.25 μm) was desalted by applying 20 μl of sample to a solid phase extraction pipette tip (TopTip, Glygen Corp.) containing 30 μlof Poros20 reverse phase C18-polymer resin (Applied Biosystems). The sample was washed four times with 100 μl of 10% MeOH in H2O (without buffer). The mitoNEET27–108 protein was eluted using four 50-μl aliquots of 50% MeOH. The eluted protein was injected on the Hewlett-Packard 5989 electrospray mass spectrometer (Agilent) with 50% MeOH as the flow injection solvent. Aliquots 2 and 3 gave the maximum recovery by mass spec abundance. These fractions were combined and divided again, and one was acidified with formic acid (2% final concentration). Approximately 1 min after acidification, the mass spectrum was acquired. Analysis of Prokaryotic Genomes—Amino acid sequences corresponding to the CDGSH domains of human mitoNEET (AAH59168) and Miner2 (EAW60525) were used to perform protein BLAST searches on the TIGR/CMR web site. The genomic organization of the prokaryotic protein hits were compared using the TIGR Genome Region Comparison feature to identify operons or gene clusters containing CDGSH domain (CD 47973) proteins. Domain composition of proteins was evaluated using the National Center for Biotechnology Information (NCBI) Conserved Domain Search. mitoNEET Binds a Redox-active 2Fe-2S Cluster—The outer mitochondrial membrane protein mitoNEET is the charter member of a small family of proteins containing a 39-aminoacid CDGSH domain (amino acids 55–93 in mitoNEET, Fig. 1A) (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). Although the CDGSH domain has been annotated as a zinc finger in the NCBI data base, our results indicated that the mitoNEET protein did not contain zinc as expected but instead bound iron (1.6 mol of iron/mol of protein) (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar). To obtain a more discriminating view of the bound iron, the optical absorbance spectrum of Δ-mitoNEET (lacking the N-terminal hydrophobic, membrane-localizing portion of the protein, Fig. 1A) was measured. The MBP-His-mitoNEET27–108 and the untagged mitoNEET33–108 proteins displayed identical spectral properties in the visible region. For simplicity, they will be collectively referred to as Δ-mitoNEET; however, details about the protein constructs used for individual experiments are presented under “Experimental Procedures.” The Δ-mitoNEET spectrum had peaks at 458 and 530 nm (Fig. 1B). This pattern resembled that of several types of 2Fe-2S cluster-containing proteins, such as ferredoxin (13Fish A. Lebendiker M. Nechushtai R. Livnah O. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 734-736Crossref PubMed Scopus (7) Google Scholar) and the Rieske Fe-S protein; (14Fee J.A. Findling K.L. Yoshida T. Hille R. Tarr G.E. Hearshen D.O. Dunham W.R. Day E.P. Kent T.A. Munck E. J. Biol. Chem. 1984; 259: 124-133Abstract Full Text PDF PubMed Google Scholar) (Fig. 1B). This result was the initial indication that the mitoNEET protein may contain an Fe-S cluster. Because many Fe-S cluster-containing proteins are redoxactive and function as electron transfer proteins (15Rees D.C. Howard J.B. Science. 2003; 300: 929-931Crossref PubMed Scopus (192) Google Scholar), we tested whether Δ-mitoNEET could be reduced/oxidized in vitro. The absorption spectra exhibited changes expected for the reduction of a 2Fe-2S center (14Fee J.A. Findling K.L. Yoshida T. Hille R. Tarr G.E. Hearshen D.O. Dunham W.R. Day E.P. Kent T.A. Munck E. J. Biol. Chem. 1984; 259: 124-133Abstract Full Text PDF PubMed Google Scholar). The peak at 458 nm showed a decrease of ∼90% in the presence of the reducing dithionite (Fig. to in recovery of the 458 nm indicating (Fig. The spectral changes that Δ-mitoNEET contained an Fe-S cluster that To the of the bound Fe-S cluster, the EPR spectrum of Δ-mitoNEET was in the oxidized and reduced states (Fig. The of a in the oxidized that the Fe-S cluster has an of iron with that to a with The spectrum observed in the dithionite-reduced protein is of a 2Fe-2S cluster with a single electron (Fig. although results could be from a cluster R. PubMed Scopus Google Scholar). data demonstrate that Δ-mitoNEET a 2Fe-2S cluster or a cluster that is To the of the iron and of the cluster, we the of mass We also of the that the Fe-S cluster of mitoNEET was acidified The mass spectra of Δ-mitoNEET exhibited a peak at (Fig. This is with the amino acid sequence of mitoNEET and the presence of a bound containing iron and acidification, the peak at and a peak at (Fig. The in to that expected for the of a single 2Fe-2S cluster. results showed that Δ-mitoNEET bound a single 2Fe-2S cluster. The CDGSH Domain Binds the 2Fe-2S with an Cys-3–His-1 2Fe-2S cluster is bound to a protein via four ligands (15Rees D.C. Howard J.B. Science. 2003; 300: 929-931Crossref PubMed Scopus (192) Google Scholar). The are in ferredoxin and the Rieske proteins. To the in Δ-mitoNEET that the 2Fe-2S cluster, we generated proteins, each with a single to and (Fig. each the recombinant protein visible and the absorbance in the nm to the 2Fe-2S cluster was (data not as The for the amino acid ligand were and in CDGSH domains (Fig. Δ-mitoNEET and were The H87Q protein visible color and optical of a bound 2Fe-2S cluster, whereas the protein exhibited an absorption spectrum to recombinant Δ-mitoNEET with a spectral of a 2Fe-2S cluster (Fig. The of iron in the H87Q and presence of iron/mol of protein in the were by analysis (data not shown). These results indicated that and were the ligands of the 2Fe-2S cluster in Δ-mitoNEET. of of the 2Fe-2S of that the 2Fe-2S cluster absorbance was under conditions to the stability of the Δ-mitoNEET protein as a function of The stability of the 2Fe-2S cluster was by the absorbance and nm. the integrity of the cluster was at 458 the peak to the mitoNEET 2Fe-2S cluster. recombinant Δ-mitoNEET was at pH there was change in the absorption spectrum with time (data not shown). the 2Fe-2S cluster of Δ-mitoNEET was at a pH of (Fig. the pH the loss of the cluster, as monitored by a change in the absorption at 458 nm (Fig. The 2Fe-2S cluster was at pH compared with pH (Fig. The of loss of the 458 nm absorption peak was at pH (data not indicating that a with a is for the stability of the cluster. To the unusual lability of the 2Fe-2S cluster, we compared the stability of which contains a Cys-3–His-1 to that of a which has a At pH 50% of the 2Fe-2S of Δ-mitoNEET was 30 min. We refer to this as the of the cluster. a of min was measured for ferredoxin (Fig. We thought this in with to was due to Δ-mitoNEET an amino acid other than as the ligand to binding of the 2Fe-2S cluster. Both and are in CDGSH domains To whether of amino acids was involved in the pH lability of the cluster, we monitored the stability of the 2Fe-2S cluster in the of Δ-mitoNEET and in a Both the and the proteins bound iron at a to the type and had a spectral of a protein containing a 2Fe-2S cluster (Fig. The demonstrated a pH from that of the wild-type Δ-mitoNEET protein (Fig. the in a change in the at pH from min for wild-type to min for the (Fig. the CDGSH domain of Δ-mitoNEET to a more center in a protein with stability of its 2Fe-2S cluster at low This indicated that protonation of upon release of the 2Fe-2S cluster. To the of our there are other of and 2Fe-2S clusters coordinated by Cys-3–His-1 both Miner1 and Miner2 the same CDGSH motif, suggesting that all members of this family are to have the 2Fe-2S clusters biophysical experiments with recombinant Miner1 and which are also in revealed absorbance dithionite and cluster lability that were with of mitoNEET (data not shown). The that CDGSH domains are from bacteria to that this unique coordination sphere for the 2Fe-2S cluster has been for of of is an drug used to treat type 2 diabetes J.R. Biochem. Pharmacol. 2006; 72: 125-131Crossref PubMed Scopus (43) Google Scholar), and mitoNEET was originally identified as a binding target of The protein of more than a mitochondrial targeting sequence with a and a 2Fe-2S-containing CDGSH domain. it is to that the of this domain in will that of mitoNEET in the mitochondria from mitoNEET-null have reduced oxidative capacity (12Wiley S.E. Murphy A.N. Ross S.A. van der Geer P Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 5318-5323Crossref PubMed Scopus (220) Google Scholar), although the by which mitoNEET this is are by its biophysical that mitoNEET may function in Fe-S cluster and/or as a redox protein. The of mitoNEET to release its Fe-S cluster is and a role for mitoNEET in Fe-S cluster and/or shuttling Fe-S clusters of the The to the protonation of a for cluster Although we by the pH in we that in protein or cofactor could the Although many of the proteins involved in the and shuttling of 2Fe-2S clusters are it is that several mitochondrial to be R. R. A. D.J. O. E. U. 2006; PubMed Scopus Google Scholar). It is that mitoNEET, the first identified 2Fe-2S protein localized to the outer mitochondrial in the 2Fe-2S cluster of mitoNEET is suggesting that it a role in redox To evidence that the CDGSH domain may function in redox we the of domain fusion and This for proteins or domains of in of M. D. Science. PubMed Scopus Google Scholar). domains into a single protein in are in proteins in prokaryotic in operons or clusters and/or There are prokaryotic proteins to and to Miner2 containing a single CDGSH domain or CDGSH of the CDGSH proteins are in gene clusters with sequences annotated as there are at of and γ having CDGSH domains to a domain (Fig. This domain is to the domain of Fig. is that such for proteins, which in this a role in redox reactions. it that in the CDGSH domain has been in this fusion protein with several other redox a or domain of which are that bind redox-active Fe-S (Fig. A. 2004; PubMed Scopus Google Scholar). one that the and Miner2 function to prokaryotic proteins, it is that mitoNEET and other members of the family have it is to that mitoNEET was originally identified by with a (11Colca J.R. McDonald W.G. Waldon D.J. Leone J.W. Lull J.M. Bannow C.A. Lund E.T. Mathews W.R. Am. J. Physiol. 2004; 286 (–E260): E252Crossref PubMed Scopus (270) Google Scholar). it is to that the with the drug could the redox or the function of the CDGSH domain of We are studies to the of mitoNEET, as well as its in function and its role in R. N. the for the We for the spectrum of the Rieske Fe-S protein from for in the EPR and for critical of the and and for with