Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p
2009; Elsevier BV; Volume: 284; Issue: 42 Linguagem: Inglês
10.1074/jbc.m109.021113
ISSN1083-351X
Autores Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoErv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX16C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur. Erv1p is a FAD-dependent sulfhydryl oxidase of the mitochondrial intermembrane space. It contains three conserved disulfide bonds arranged in two CXXC motifs and one CX16C motif. Experimental evidence for the specific roles of the individual disulfide bonds is lacking. In this study, structural and functional roles of the disulfides were dissected systematically using a wide range of biochemical and biophysical methods. Three double cysteine mutants with each pair of cysteines mutated to serines were generated. All of the mutants were purified with the normal FAD binding properties as the wild type Erv1p, showing that none of the three disulfides are essential for FAD binding. Thermal denaturation and trypsin digestion studies showed that the CX16C disulfide plays an important role in stabilizing the folding of Erv1p. To understand the functional role of each disulfide, small molecules and the physiological substrate protein Mia40 were used as electron donors in oxygen consumption assays. We show that both CXXC disulfides are required for Erv1 oxidase activity. The active site disulfide is well protected thus requires the shuttle disulfide for its function. Although both mutants of the CXXC motifs were individually inactive, Erv1p activity was partially recovered by mixing these two mutants together, and the recovery was rapid. Thus, we provided the first experimental evidence of electron transfer between the shuttle and active site disulfides of Erv1p, and we propose that both intersubunit and intermolecular electron transfer can occur. Disulfide bonds play very important roles in the structure and function of many proteins by stabilizing protein folding and/or acting as thiol/disulfide redox switches. The process of disulfide formation is catalyzed by dedicated enzymes in vivo (1Collet J.F. Bardwell J.C. Mol. Microbiol. 2002; 44: 1-8Crossref PubMed Scopus (176) Google Scholar, 2Kadokura H. Katzen F. Beckwith J. Annu. Rev. Biochem. 2003; 72: 111-135Crossref PubMed Scopus (446) Google Scholar, 3Sevier C.S. Kaiser C.A. Antioxid. Redox. Signal. 2006; 8: 797-811Crossref PubMed Scopus (91) Google Scholar, 4Stojanovski D. Müller J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Biochim. Biophys. Acta. 2008; 1783: 610-617Crossref PubMed Scopus (46) Google Scholar). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4Stojanovski D. Müller J.M. Milenkovic D. Guiard B. Pfanner N. Chacinska A. Biochim. Biophys. Acta. 2008; 1783: 610-617Crossref PubMed Scopus (46) Google Scholar, 5Lee J. Hofhaus G. Lisowsky T. FEBS Lett. 2000; 477: 62-66Crossref PubMed Scopus (155) Google Scholar, 6Koehler C.M. Tienson H.L. Biochim. Biophys. Acta. 2009; 1793: 139-145Crossref PubMed Scopus (49) Google Scholar). It is an essential component of the redox regulated Mia40/Erv1 import and assembly pathway used by many of the cysteine-containing intermembrane space proteins, such as members of the "small Tim" and Cox17 families (7Mesecke N. Terziyska N. Kozany C. Baumann F. Neupert W. Hell K. Herrmann J.M. Cell. 2005; 121: 1059-1069Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 8Tokatlidis K. Cell. 2005; 121: 965-967Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 9Allen S. Balabanidou V. Sideris D.P. Lisowsky T. Tokatlidis K. J. Mol. Biol. 2005; 353: 937-944Crossref PubMed Scopus (193) Google Scholar, 10Hell K. Biochim. Biophys. Acta. 2008; 1783: 601-609Crossref PubMed Scopus (86) Google Scholar). Upon import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate. Although oxidized Mia40 promotes disulfide bond formation in the substrates, Erv1p functions in catalyzing reoxidation of the reduced Mia40 and/or release of the substrate (11Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 12Banci L. Bertini I. Cefaro C. Ciofi-Baffoni S. Gallo A. Martinelli M. Sideris D.P. Katrakili N. Tokatlidis K. Nat. Struct. Mol. Biol. 2009; 16: 198-206Crossref PubMed Scopus (210) Google Scholar, 13Stojanovski D. Milenkovic D. Müller J.M. Gabriel K. Schulze-Specking A. Baker M.J. Ryan M.T. Guiard B. Pfanner N. Chacinska A. J. Cell Biol. 2008; 183: 195-202Crossref PubMed Scopus (71) Google Scholar). The common features for the FAD-dependent sulfhydryl oxidases are that the enzymes can catalyze the electron transfer from substrate molecules (e.g. protein thiols) through the noncovalent bound FAD cofactor to molecular oxygen or oxidized cytochrome c (14Thorpe C. Coppock D.L. J. Biol. Chem. 2007; 282: 13929-13933Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The sulfhydryl oxidases can be divided into three groups: Ero1 enzymes, multidomain quiesin sulfhydryl oxidases, and single domain Erv (essential for respiration and vegetative growth)/ALR proteins. The yeast Ero1p and the mammalian homologues (Ero1α and Ero1β) are large flavoenzymes present in the ER with at least five disulfide bonds, but only two of the disulfide bonds are conserved. The conserved cysteines are essential for the catalytic activity of Ero1p forming the active site CXXC and shuttle disulfide CX4C, respectively (15Frand A.R. Cuozzo J.W. Kaiser C.A. Trends Cell Biol. 2000; 10: 203-210Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, 16Gross E. Kastner D.B. Kaiser C.A. Fass D. Cell. 2004; 117: 601-610Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). Furthermore, nonconserved disulfide bonds have been shown recently to be important in regulating the activity of both yeast and mammalian Ero1 (17Sevier C.S. Qu H. Heldman N. Gross E. Fass D. Kaiser C.A. Cell. 2007; 129: 333-344Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 18Baker K.M. Chakravarthi S. Langton K.P. Sheppard A.M. Lu H. Bulleid N.J. EMBO J. 2008; 27: 2988-2997Crossref PubMed Scopus (116) Google Scholar, 19Appenzeller-Herzog C. Riemer J. Christensen B. Sørensen E.S. Ellgaard L. EMBO J. 2008; 27: 2977-2987Crossref PubMed Scopus (144) Google Scholar). The second group of oxidases, the multidomain quiesin sulfhydryl oxidases, have important functions in higher eukaryotes (14Thorpe C. Coppock D.L. J. Biol. Chem. 2007; 282: 13929-13933Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 20Coppock D.L. Thorpe C. Antioxid. Redox. Signal. 2006; 8: 300-311Crossref PubMed Scopus (82) Google Scholar). Quiesin sulfhydryl oxidases consist of an Erv/ALR module fused to one or more thioredoxin-like domains with two conserved CXXC motifs in the Erv/ALR module. Quiesin sulfhydryl oxidase enzymes are found in many subcellular and extracellular locations, but not in mitochondria. Instead, single domain Erv/ARL enzymes of the third group are found in the 7mitochondria of many eukaryotic cells (21Fass D. Biochim. Biophys. Acta. 2008; 1783: 557-566Crossref PubMed Scopus (95) Google Scholar). Erv1p belongs to this single domain Erv/ARL family, which includes the human mitochondrial ARL, plant AtErv1, and yeast Erv2p of the ER lumen. The Erv/ARL enzymes are characterized by a highly conserved central catalytic core of ∼100 amino acids, which includes an active site CXXC motif (Cys130–Cys133 for Erv1p), CX16C disulfide bond (Cys159–Cys176 for Erv1p), and residues involved in FAD binding (Fig. 1A). Based on the partial crystal structure data of Erv2p (22Gross E. Sevier C.S. Vala A. Kaiser C.A. Fass D. Nat. Struct. Biol. 2002; 9: 61-67Crossref PubMed Scopus (163) Google Scholar) and AtErv1 (23Vitu E. Bentzur M. Lisowsky T. Kaiser C.A. Fass D. J. Mol. Biol. 2006; 362: 89-101Crossref PubMed Scopus (68) Google Scholar), the catalytic core of Erv proteins contains a four-helix bundle forming the noncovalent FAD-binding site with the active site CXXC in close proximity to the isoalloxazine ring of FAD. In addition, the long range CX16C disulfide bond of the Erv proteins brings the short fifth helix to the four-helix bundle in proximity to the adenine ring of FAD (Fig. 1A). Thus, the CX16C disulfide bond is proposed to play a structural role in stabilizing the FAD binding and/or protein folding, but direct experimental evidence to verify the roles is lacking. Apart from the catalytic core, the other parts of the proteins seem flexible and unfolded. Importantly, all members of the Erv/ALR family have at least an additional disulfide bond located in the nonconserved N- or C-terminal region to the catalytic core (Fig. 1B), which is hypothesized as a shuttle disulfide based on the partial crystal structure of Erv2 (22Gross E. Sevier C.S. Vala A. Kaiser C.A. Fass D. Nat. Struct. Biol. 2002; 9: 61-67Crossref PubMed Scopus (163) Google Scholar). The hypothesized shuttle disulfide of Erv2p CXC and AtErv1 CX4C are located in the C terminus, but Erv1p (Cys30–Cys33) and ALR have a CXXC shuttle disulfide located N-terminal to the catalytic core. Furthermore, structural and chemical data have suggested that Erv/ARL enzymes form homodimer or oligomers in the presence or absence of intermolecular disulfide bonds (5Lee J. Hofhaus G. Lisowsky T. FEBS Lett. 2000; 477: 62-66Crossref PubMed Scopus (155) Google Scholar, 23Vitu E. Bentzur M. Lisowsky T. Kaiser C.A. Fass D. J. Mol. Biol. 2006; 362: 89-101Crossref PubMed Scopus (68) Google Scholar, 24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). Yeast mitochondrial Erv1p contains a total of six Cys residues forming three pairs of disulfide bonds (residues 30–33, 130–133, and 159–176) as described above. Previous studies with single Cys mutants showed that although all three disulfide bonds are essential for Erv1p function in vivo, only Cys130–Cys133 disulfide is required for the oxidase activity of Erv1p in vitro (24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). The conclusion that only Cys130–Cys133 disulfide is required for Erv1p oxidase activity in vitro was based on a study using the artificial substrate DTT 2The abbreviations used are: DTT1,4-dithiothreitolAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidTCEPtris(2-carboxyethyl)phosphineWTwild typeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePKproteinase K. 2The abbreviations used are: DTT1,4-dithiothreitolAMS4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acidTCEPtris(2-carboxyethyl)phosphineWTwild typeTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycinePKproteinase K. as the electron donor. Abnormal color changes were observed for some of the single Cys mutants of Erv1p in the previous study that were probably caused by protein misfolding or formation of non-native disulfides because of the presence of a redox active but unpaired Cys. It is clear that Cys130–Cys133 is the active site disulfide; however, experimental evidence for the role of Cys30–Cys33 disulfide is lacking, and the specific role played by the unique CX16C motif of Erv proteins is unknown. 1,4-dithiothreitol 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid tris(2-carboxyethyl)phosphine wild type N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine proteinase K. 1,4-dithiothreitol 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid tris(2-carboxyethyl)phosphine wild type N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine proteinase K. In this study, we dissected the structural and functional roles of all three individual disulfides of Erv1p systematically. To avoid misfolding via unpaired Cys, three double Cys mutants of Erv1p were generated with each of the disulfides mutated to serines. All three mutants were successfully purified with the normal FAD binding properties of the wild type (WT) Erv1p. Various biophysical and biochemical methods were used to study the folding and oxidase activity of the WT and Erv1p mutants. Both artificial and the natural substrate (Mia40) of Erv1p were used as electron donors to understand the functional mechanism of Erv1p. Our results show that both the first (Cys30–Cys33) and second (Cys130–Cys133) disulfides are essential for Erv1 oxidase activity in vitro. Although none of the three disulfides are essential for FAD binding, the third disulfide (Cys159–Cys176) plays an important role in stabilizing the folding of Erv1p. More importantly, this study provided direct experimental evidence to show that Cys30–Cys33 functionally acts as a shuttle disulfide passing electrons to the active site Cys130–Cys133 disulfide. Moreover, the electron transfer seems to occur through both intersubunit and intermolecular interactions. 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Molecular Probes (Invitrogen). EDTA was from BDH Co, and all other chemicals were obtained from Sigma at the highest grade. A peptide corresponding to 14 C-terminal residues of Erv1p was used to raise antibodies in rabbit against Erv1p (Eurogentec Ltd.). Cysteine to serine mutants of Erv1 were created using QuikChange site-directed mutagenesis with Pfu DNA polymerase (Stratagene) and pET-24a(+) harboring the wild type complete Erv1 gene as template (5Lee J. Hofhaus G. Lisowsky T. FEBS Lett. 2000; 477: 62-66Crossref PubMed Scopus (155) Google Scholar). All of the constructs were verified by DNA sequencing. Sequences of mutagenic oligonucleotides can be provided upon request. The Erv1p-His6 proteins were expressed in the Escherichia coli Rosetta-gamiTM 2 (Novagen) and purified using His tag affinity beads followed by fast protein liquid chromatography using Superdex75 column as described previously (25Morgan B. Ang S.K. Yan G. Lu H. J. Biol. Chem. 2009; 284: 6818-6825Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Concentrations of the WT and Erv1p mutants were calculated using the molar extinction coefficients determined in this study as listed in Table 1. Mia40c (amino acids 284–403), the C-terminal domain of Mia40, was cloned into pGEX 4T-1 vector (GE Healthcare), expressed in the E. coli Rosetta-gamiTM 2, and purified using GST affinity beads as described previously (11Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The partially reduced Mia40c (Mia40c-pR) was prepared by incubation with 0.5 mm TCEP for 20 min at room temperature, followed by gel filtration using a Superdex75 column to remove TCEP. The protein had the same redox state as that described in Ref. 11Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar. The concentration was determined based on a 5,5′-dithiobis(nitrobenzoic acid) assay for the free thiol groups. All of the experiments were carried out under aerobic conditions at 25 °C in buffer AE (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA), unless indicated otherwise.TABLE 1Oxygen consumption kinetic parameters for the WT and Erv1p mutantsElectron donorErv1pkcatKmkcat/Kms−1mmm−1 s−110 mm DTTWT1.3 ± 0.157 ± 42.3 ± 0.2 × 104C30S/C33S1.5 ± 0.162 ± 52.4 ± 0.2 × 104C130S/C133S<0.1C159S/C176S0.8 ± 0.187 ± 89.2 ± 0.2 × 1033.5 mm TCEPWT1.1 ± 0.127 ± 34.1 ± 0.3 × 104C30S/C33S<0.1C130S/C133S<0.05C159S/C176S0.7 ± 0.118 ± 23.9 ± 0.3 × 104 Open table in a new tab At various time points, protein aliquots were removed from reaction solutions and added to nonreducing gel sample buffer containing excess amount of AMS (10 mm) for 30 min in the dark at room temperature as described before (26Lu H. Allen S. Wardleworth L. Savory P. Tokatlidis K. J. Biol. Chem. 2004; 279: 18952-18958Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). AMS interacts with free thiols of reduced proteins covalently, but not disulfide bonds. Each bound AMS molecule increases the molecular mass of the protein ∼0.5 kDa. Different redox states of the proteins were analyzed by 16% Tricine-SDS-PAGE under nonreducing conditions. Absorption spectra of Erv1p and its mutants were recorded using a Cary 300 spectrophotometer from 250 to 700 nm, at 1-nm intervals, using a 1-cm path length quartz cuvette. The extinction coefficients and the percentage of enzyme-bound FAD for the WT and mutant Erv1p were calculated based on a molar extinction coefficient of 11.3 mm−1 cm−1 at 450 nm for free FAD and 72.68 mm−1 cm−1 for Erv1p at 275 nm as reported previously (27Dabir D.V. Leverich E.P. Kim S.K. Tsai F.D. Hirasawa M. Knaff D.B. Koehler C.M. EMBO J. 2007; 26: 4801-4811Crossref PubMed Scopus (137) Google Scholar). FAD was released from the proteins by the addition of 1% SDS. CD analysis was performed using a JASCO J810 spectropolarimeter with a 1-mm path length quartz cuvette. Far-UV CD spectra were measured at 25 °C with 300 μl of 10 μm proteins as described previously (28Lu H. Woodburn J. J. Mol. Biol. 2005; 353: 897-910Crossref PubMed Scopus (47) Google Scholar). Each spectrum represents an average of four scans from 200 to 260 nm at 0.2-nm intervals with the spectra for buffer alone subtracted. Thermal denaturation was measured at 222 nm, at 1 °C intervals over 5–90 °C with temperature increase of 1 °C/min. 20 μl of 5 μm Erv1p and its mutants were incubated with 50 μg/ml proteinase K at 25 °C for 30 min, followed by inhibition of the protease activity by the addition of 10 mm phenylmethylsulfonyl fluoride for 10 min. Then the samples were analyzed by 16% Tricine-SDS-PAGE and Western blotting with antibody raised with peptide of the C terminus of Erv1p. Mock controls were treated in exactly the same manner. Oxygen consumption of Erv1 was measured using a Clarke-type oxygen electrode (Hansatech Instrument Ltd.) at 25 °C as described before (25Morgan B. Ang S.K. Yan G. Lu H. J. Biol. Chem. 2009; 284: 6818-6825Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). For measurements with DTT and TCEP as electron donors, 1 or 2 μm Erv1 as indicated in the text was pre-equilibrated at 25 °C followed by the addition of 10 mm DTT or 3.5 mm TCEP to initiate the reaction. For measurements with Mia40c-pR as substrate, 50 μm freshly prepared Mia40c-pR was pre-equilibrated at 25 °C followed by the addition of 1 μm Erv1 to catalyze the reaction. The WT Erv1 (5 μm) was incubated with 0 or 50 μm freshly prepared Mia40c-pR for ∼10 s, and then the reaction was stopped by the addition of nonreducing SDS-PAGE sample buffer containing 4 mm iodoacetamide. The proteins were separated by nonreducing SDS-PAGE. The bands corresponding to Erv1 were excised and digested with AspN. The peptides were analyzed by mass spectrometry on a Bruker matrix-assisted laser desorption ionization time-of-flight using a positive reflection method. To understand the roles of individual disulfide bonds of Erv1, three double Cys mutants of Erv1p with the Cys residues corresponding to each of the three disulfides mutated to serines were generated. They are named as C30S/C33S, C130S/C133S, and C159S/C176S, respectively, in the rest of the report. All three mutants were successfully purified with the same method and yellowish color as that of the WT Erv1p. No abnormal color was observed for any of the mutants. The UV-visible spectrum of the WT Erv1p shows a characteristic bound FAD spectrum with a maximum absorbance at 460 nm and a shoulder peak at ∼485 nm (supplemental Fig. S1). The absorption maximum was ∼10 nm blue-shifted to 450 nm upon the addition of 1% SDS (data not shown), the same wavelength as that of free FAD confirming the release of cofactor FAD. The same protein-bound FAD spectrum as that of the WT Erv1p was observed for C30S/C33S and C159S/C176S mutants (supplemental Fig. S1), but a slightly blue-shifted spectrum with the maximum at 453 nm was obtained for C130S/C133S (supplemental Table S1). It is consistent with the fact that the active site Cys130–Cys133 disulfide is located proximal to the isoalloxazine ring of FAD and the mutation changes bound-FAD absorption slightly. The molar extinction coefficients for the bound FAD in the WT and all three double Cys mutants were determined (see "Experimental Procedures") to be 11.9, 11.1, 12.1, and 11.9 mm−1 cm−1 at the corresponding wavelength of the absorption maximum (supplemental Table S1). These values are similar to each other and to that of other members of Erv/ALR family. The same FAD-binding yield of ∼93% was obtained for the WT and all the mutants. Taken together, these results show that all three double Cys mutants were correctly folded and with FAD bound at a molar ratio of 1:1 as that of the WT. None of the three individual disulfide bonds of Erv1p is essential for FAD binding. It was shown that Cys30 and Cys33 are involved in formation of an intermolecular disulfide bonded dimer and oligomers (5Lee J. Hofhaus G. Lisowsky T. FEBS Lett. 2000; 477: 62-66Crossref PubMed Scopus (155) Google Scholar, 24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). Thus, the oligomerization state of the double Cys mutants was investigated using reducing and nonreducing SDS-PAGE. For all the proteins except C30S/C33S mutant, a fraction of ∼20% proteins migrated slowly on the nonreducing gel with an apparent molecular weight corresponding to a dimer (data not shown). The result is consistent with the previous observation (5Lee J. Hofhaus G. Lisowsky T. FEBS Lett. 2000; 477: 62-66Crossref PubMed Scopus (155) Google Scholar, 24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). Next, the effect of these mutations on the overall conformation of Erv1p was investigated using far-UV CD spectra. The WT and all of the mutants have a similar spectrum profile with a conformation dominated by α-helical structures as expected (data not shown). However, an intensity decrease was observed for C30S/C33S mutant. It may be due to the absence of intermolecular disulfide bonded dimer, or the Cys30–Cys33 disulfide may be important for the overall folding of the non-FAD-binding N-terminal segment. To understand the possible structural role played by each disulfide, thermal denaturation of the WT and Erv1p mutants was studied using CD at 222 nm. As shown in Fig. 2A, the WT Erv1p is stable against heat denaturation with a melting temperature (Tm) at ∼68 °C. The N-terminal double Cys mutant (C30S/C33S) had no apparent affect on the overall stability of Erv1p. In contrast, both the core domain double Cys mutants (C130S/C133S and C159S/C176S) had a clear effect on the stability of Erv1p, with a Tm of 52 °C for C130S/C133S and 38 °C for C159S/C176S (Fig. 2A). Mutation of the Cys159-Cys167 disulfide alone resulted in a decrease of 30 °C in Erv1p Tm. A fraction of the C159S/C176S mutant was unfolded at the physiological temperature and as low as ∼25 °C. Thus, our results show that the Cys159–Cys176 disulfide plays a key role in stabilizing the overall folding of Erv1p. Next, the effects of the individual disulfides on the stability of Erv1 were confirmed by proteinase K (PK) digestion analysis (Fig. 2B). After incubation of the WT and the mutants with or without the presence of 50 μg/ml PK at 25 °C for 30 min, the samples were analyzed by Western blotting using antibody against the C terminus of Erv1p. In the presence of PK, the WT and all three double Cys mutants were degraded (Fig. 2B). Although a stable C-terminal fragment of ∼15 kDa was clearly observed for the WT and C30S/C33S mutant, the intensity of the same fragment was very weak for C130S/C133S and C159S/C176S mutants, and no other bands were detectable. Thus, the results of PK digestion are consistent with those of the thermal denaturation study. Taken together, CD and PK digestion studies show that the C-terminal region of Erv1p was folded and resistant to PK digestion but not the N terminus. Therefore, although the N-terminal disulfide Cys30–Cys33 has no effect on the stability of Erv1p, both of the central core disulfides play a role in stabilizing the folding of Erv1p, especially the Cys159–Cys176 disulfide. The effects of individual disulfide bonds on the sulfhydryl oxidase activity of Erv1p were studied using oxygen consumption assays. First, the commonly used reducing agent DTT was employed as the electron donor with and without the presence of the WT or mutant Erv1p. As shown in Fig. 3A, oxygen consumption was catalyzed as soon as the WT Erv1p was added. The kcat was determined to be 1.3 ± 0.1 s−1, ∼50% higher than that reported previously (24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar, 27Dabir D.V. Leverich E.P. Kim S.K. Tsai F.D. Hirasawa M. Knaff D.B. Koehler C.M. EMBO J. 2007; 26: 4801-4811Crossref PubMed Scopus (137) Google Scholar). The Km for molecular oxygen was determined to be 57 μm (Fig. 3B and Table 1). Different effects on the oxidase activity were observed with the three double Cys mutants. As expected, the active site C130S/C133S mutant showed no or very little activity, similar to that of a previous study using single Cys mutants (24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). Interestingly, the N-terminal C30S/C33S mutant showed ∼15% higher oxidase activity (kcat = 1.5 ± 0.1 s−1) than that of the WT enzyme. In contrast, the previous study with the corresponding single Cys mutants showed only ∼30–50% activity of the WT Erv1p (24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). For the C159S/C176S mutant, a decreased activity was observed (Fig. 3A), and the kcat and Km values were determined to be 0.8 s−1 and 87 μm, respectively (Table 1). The enzyme specificity, ratio of kcat/Km, was the same (2.3 × 104 m−1 s−1) for the WT and C30S/C33S and was similar to that of Ero1 proteins (4–8 × 104 m−1 s−1) (29Gross E. Sevier C.S. Heldman N. Vitu E. Bentzur M. Kaiser C.A. Thorpe C. Fass D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 299-304Crossref PubMed Scopus (293) Google Scholar, 30Wang L. Li S.J. Sidhu A. Zhu L. Liang Y. Freedman R.B. Wang C.C. J. Biol. Chem. 2009; 284: 199-206Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Taken together, these results showed that using DTT as substrate, only Cys130–Cys133 disulfide is required for the oxidase activity of Erv1p, confirming that it is the active site disulfide. Previous yeast genetic studies demonstrated that all six Cys residues of Erv1p were required for its function in vivo (24Hofhaus G. Lee J.E. Tews I. Rosenberg B. Lisowsky T. Eur. J. Biochem. 2003; 270: 1528-1535Crossref PubMed Scopus (71) Google Scholar). Therefore, we asked whether all three disulfides are essential for the oxidase active toward its native substrate protein Mia40. To this end, a functional C-terminal domain of Mia40, Mia40c (residues 284–403), was expressed and purified as reported previously. Mia40c contains all the six conserved Cys residues (CPC-CX9C-CX9C) of the protein. It has been shown that the CPC motif is the redox active site of Mia40, which can be selectively reduced and act as an electron donor for Erv1p (11Grumbt B. Stroobant V. Terziyska N. Israel L. Hell K. J. Biol. Chem. 2007; 282: 37461-37470Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Thus, the partially reduced Mia40c (Mia40c-pR), with the Cys of CPC in the reduced form and CX9C motifs in the oxidized form, was prepared and used as an electron donor for Erv1p. Oxygen consumption of 50 μm Mia40c-pR in the presence of 1 μm the WT or mutant Erv1p was measured. As shown in Fig. 4A, whereas both the WT and C159S/C176S Erv1p can catalyze the electron transfer from Mia40 to molecular oxygen, both C30S/C33S and C130S/C133S mutants were inactive (Fig. 4A). The initial rates for WT and C159S/C176S were ∼0.6 ± 0.1 and 0.5 ± 0.1 s−1, respectively. Thus, for Mia40 oxidation, although C159S/C176S mutant had no significant effect on the oxidase activity of
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