Deciphering Structural and Functional Roles of Individual Disulfide Bonds of the Mitochondrial Sulfhydryl Oxidase Erv1p

Abstract

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 (1–4). Erv1p is a FAD-dependent sulfhydryl oxidase located in the Saccharomyces cerevisiae mitochondrial intermembrane space (4–6). 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 (7–10). 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 (11–13).

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 (14). 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 (15, 16). Furthermore, nonconserved disulfide bonds have been shown recently to be important in regulating the activity of both yeast and mammalian Ero1 (17–19). The second group of oxidases, the multidomain quiesin sulfhydryl oxidases, have important functions in higher eukaryotes (14, 20). 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 (21). 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 (22) and AtErv1 (23), 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 (22). 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 (5, 23, 24).

FIGURE 1.

Structure and conserved Cys motifs of Erv/ALR enzymes. A, modeled structures of the conserved central catalytic core domain of Erv1p dimer based on the crystal structure data of AtErv1 (Protein Data Bank accession number 2HJ3, residues 73–173, the helix 1 starts with residue 75). The helices of the four-helix bundle and the short fifth helix are labeled from 1 to 5. The two disulfides are shown as yellow spheres, and the cofactor FAD is in red. The Cys¹³⁰–Cys¹³³ is the redox active disulfide located closely to the isoalloxazine ring of FAD. The N and C termini were labeled as N and C, respectively. The structure was generated using Pymol program. B, schematic of the primary structure of yeast, plant, and human sulfhydryl oxidase with the conserved Cys motifs. The conserved central catalytic core regions are shown as black bars, and the nonconserved regions are in gray.

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 (24). The conclusion that only Cys¹³⁰–Cys¹³³ disulfide is required for Erv1p oxidase activity in vitro was based on a study using the artificial substrate DTT² 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.

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.

EXPERIMENTAL PROCEDURES

Materials

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.).

Mutagenesis and Protein Preparations

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 (5). 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-gami^TM 2 (Novagen) and purified using His tag affinity beads followed by fast protein liquid chromatography using Superdex75 column as described previously (25). 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-gami^TM 2, and purified using GST affinity beads as described previously (11). 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. 11. 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 1.

Oxygen consumption kinetic parameters for the WT and Erv1p mutants

All of the experiments were carried out with 1 mm Erv1p proteins in 50 mm Tris buffer, pH 7.4, 150 mm NaCl, 1 mm EDTA at 25 °C. The error bars represent the standard errors (n = 3).

Electron donor	Erv1p	kcat	Km	kcat/Km
		s−1	mm	m−1s−1
10 mm DTT	WT	1.3 ± 0.1	57 ± 4	2.3 ± 0.2 × 104
	C30S/C33S	1.5 ± 0.1	62 ± 5	2.4 ± 0.2 × 104
	C130S/C133S	<0.1
	C159S/C176S	0.8 ± 0.1	87 ± 8	9.2 ± 0.2 × 103
3.5 mm TCEP	WT	1.1 ± 0.1	27 ± 3	4.1 ± 0.3 × 104
	C30S/C33S	<0.1
	C130S/C133S	<0.05
	C159S/C176S	0.7 ± 0.1	18 ± 2	3.9 ± 0.3 × 104

AMS Assays

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 (26). 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.

Determination of the Extinction Coefficients

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 (27). FAD was released from the proteins by the addition of 1% SDS.

Circular Dichroism

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 (28). 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.

Proteinase K Digestion

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 Assay

Oxygen consumption of Erv1 was measured using a Clarke-type oxygen electrode (Hansatech Instrument Ltd.) at 25 °C as described before (25). 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.

Mass Spectrometry Analysis

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.

RESULTS

Characterization of the WT and Three Double Cys Mutants of Erv1p

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.

Structural Roles Played by Individual Disulfide Bonds of Erv1p

It was shown that Cys³⁰ and Cys³³ are involved in formation of an intermolecular disulfide bonded dimer and oligomers (5, 24). 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 (5, 24). 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 (T_m) 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 T_m 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 T_m. 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.

FIGURE 2.

Stability of the WT and mutant Erv1p. A, thermal denaturation of the WT and Erv1p mutants measured by CD intensity change at 222 nm. The T_m of the WT Erv1p (solid circle), C30S/C33S (cross), C130S/C133S (open circle), and C159S/C176S (open triangle) mutant were determined to be 68, 67, 52, and 38 °C, respectively. B, Western blotting of Erv1p and its mutants using an antibody against the C-terminal of Erv1p. The proteins were untreated or treated by incubation with 0.05 mg/ml proteinase K at 25 °C for 30 min and followed by the addition of 10 mm phenylmethylsulfonyl fluoride before separation by SDS-PAGE. The full-length Erv1p and the N-terminal truncated fragments (*) are indicated.

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 the Individual Disulfides on the Oxidase Activity of Erv1p

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 k_cat was determined to be 1.3 ± 0.1 s⁻¹, ∼50% higher than that reported previously (24, 27). The K_m 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 (24). Interestingly, the N-terminal C30S/C33S mutant showed ∼15% higher oxidase activity (k_cat = 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 (24). For the C159S/C176S mutant, a decreased activity was observed (Fig. 3A), and the k_cat and K_m values were determined to be 0.8 s⁻¹ and 87 μm, respectively (Table 1). The enzyme specificity, ratio of k_cat/K_m, 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) (29, 30). 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.

FIGURE 3.

Oxygen consumption of DTT catalyzed by the WT and Erv1p mutants. A, oxygen consumption profiles of 10 mm DTT in the presence of 1 μm the WT (curve a), C30S/C33S (curve b), C130S/C133S (curve c), and C159S/C176S (curve d), respectively, and two controls of 10 mm DTT in the buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA) alone (curve e) or plus 1 μm free FAD (curve f). For all measurements, DTT was injected to pre-equilibrated samples at 25 °C at time 0. B, oxygen consumption of 10 mm DTT catalyzed by the WT Erv1p and the time course of the rate change (inset) to show how k_cat and K_m were determined.

Both Cys³⁰–Cys³³ and Cys¹³⁰–Cys¹³³ Disulfides Are Required for the Oxidase Activity of Erv1p toward Its Physiological Substrate

Previous yeast genetic studies demonstrated that all six Cys residues of Erv1p were required for its function in vivo (24). 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 (11). 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 Erv1p, both Cys³⁰–Cys³³ and Cys¹³⁰–Cys¹³³ disulfides were required. It seems that less oxygen was consumed (∼40 μm) than might be expected given that 50 μm Mia40 was in the reaction. This may partially be due to the fact that the protein was not 100% pure (∼95% based on SDS-PAGE), due to errors of concentration as determined by the 5,5′-dithiobis(nitrobenzoic acid) assay, and/or due to the fact that oxygen consumption was very slow after the reaction for 3 min.

FIGURE 4.

Oxygen consumption and AMS assay of Mia40c-pR oxidation catalyzed by the WT and Erv1p mutants. A, oxygen consumption profiles of 50 μm Mia40c-pR in the presence of 1 μm the WT Erv1p (curve a), C30S/C33S (curve b), C130S/C133S (curve c), and C159S/C176S (curve d), respectively. B, AMS assay of the redox state change of Mia40c-pR. Mia40c was detected by Coomassie Blue staining. C, AMS assay of the redox state change of the WT and Erv1p mutants. The proteins were detected by Western blotting with antibody against Erv1p. D, mass spectrometry analysis of Erv1p before (panel a) and after (panel b) incubated with Mia40c-pR for ∼10 s. The peptides contain Cys³⁰ and Cys³³ in the oxidized (1548.69 Da) or reduced and alkylated (1664.88 Da) forms were shown.

Furthermore, changes in the redox state of Erv1p and Mia40c were analyzed in parallel using a SDS-PAGE based AMS thiol-modification assay (see “Experimental Procedures”). As shown in Fig. 4B, a fraction of oxidized Mi40c (O) was observed after addition of the WT or C159S/C176S Erv1p for 0.5 min, and all of the proteins were oxidized in 5 min (Fig. 4B, lanes 3–5 and 12–14). When C30S/C33S or C130S/C133S mutant was used, Mia40c-pR remained in the reduced form (R), even after 20 min (Fig. 4B, lanes 6–11). Meanwhile, the redox state of Erv1p was revealed by Western blotting (Fig. 4C). Again, the same pattern of redox state change was observed for the WT Erv1p and C159S/C176S mutant (Fig. 4C, lanes 1–4 and 13–16), which was in agreement with the time courses of Mia40c-pR oxidation. A fraction of reduced Erv1p was detected at 0.5 min, which was reoxidized/regenerated in 5 min when all of the Mia40c- pR were oxidized. For C30S/C33S mutant, no redox state change was observed, consistent with the observation that this mutant was inactive in oxygen consumption (Fig. 4A, curve b). However, all C130S/C133S mutant was reduced after 0.5 min and remained in the reduced form through the same time course (Fig. 4C, lanes 10–12), although the mutant was enzymatically inactive (Fig. 4A, curve c). Thus, these results are consistent with the conclusion that Cys³⁰–Cys³³ acts as a shuttle disulfide, which was reduced by Mia40c-pR, and these two Cys residues (Cys³⁰ and Cys³³) were modified by AMS. The conclusion was confirmed by proteinase digestion coupled with mass spectrometry analysis of Erv1 before and after incubation with Mia40c-pR briefly (Fig. 4D). Although a peptide of 1548.69 Da corresponding to 24–37 residues of Erv1 with Cys³⁰–Cys³³ disulfide bonded was identified in both samples, the same peptide with both Cys³⁰ and Cys³³ modified by iodoacetamide (1664.88 Da) was observed only in the sample mixed with Mia40c-pR (Fig. 4D, panel b).

In summary, oxygen consumption and AMS assays showed that both CXXC disulfides (Cys³⁰–Cys³³ and Cys¹³⁰–Cys¹³³) are essential for Erv1p oxidase activity toward the physiological substrate Mia40c. The CX₁₆C disulfide has no obvious effect on Erv1p activity and thus is not required for Erv1p oxidase function.

The Active Site Cys¹³⁰–Cys¹³³ Disulfide Is Well Protected and Can Be Activated by Cys³⁰–Cys³³ Disulfide through Intermolecular Electron Transfer

To understand whether Cys³⁰–Cys³³ was required to act as a shuttle disulfide and whether that requirement was due to the fact that the active site disulfide was not accessible, we repeated the oxygen consumption experiments using TCEP as an electron donor. TCEP is a stronger reducing agent and has a slightly larger size (250 Da) than DTT (154 Da). Comparison between DTT and TCEP shows that the same kind of effects on the Erv1p activity were observed for C130S/C133S (inactive) and C159S/C176S (decreased activity) mutants, respectively (compare Figs. 3A and 5A). However, an opposite result was observed for the C30S/C33S mutant. Although a slightly increased activity was observed with DTT (Fig. 3A, curve b), little oxygen consumption was detected using TCEP as an electron donor (Fig. 5A, curve b). In the absence of Cys³⁰–Cys³³ disulfide, TCEP is not a substrate for Erv1p. Thus, our results show that even TCEP cannot access and reduce the active site disulfide of Erv1 directly, suggesting the active site disulfide (Cys¹³⁰–Cys¹³³) is buried and well protected. The oxygen consumption curves of TCEP oxidation were further analyzed, and the k_cat values were determined to be 1.1 and 0.7 s⁻¹ for the WT and C159S/C176S, respectively. The K_m values for oxygen were 27 μm for the WT and 18 μm for C159S/C176S (Table 1). A similar substrate specificity of 4 × 10⁴ m⁻¹ s⁻¹ was obtained, which was ∼2–4 times higher than that determined by using DTT (Table 1). Thus, TCEP is a better electron donor than DTT in terms of activation specificity. It is consistent with the finding that DTT not only can reduce Cys³⁰–Cys³³, but also Cys¹³⁰–Cys¹³³ and maybe even Cys¹⁵⁹–Cys¹⁷⁶; thus some enzyme activity may be lost during the assays using DTT.

FIGURE 5.

Oxygen consumption of TCEP catalyzed by the WT and mutant Erv1. A, oxygen consumption profiles of 3.5 mm TCEP in the presence of 1 μm the WT Erv1p (curve a), C30S/C33S (curve b), C130S/C133S (curve c), and C159S/C176S (curve d), respectively, and two controls of 3.5 mm TCEP in the buffer alone (curve e) or in the presence of 1 μm free FAD (curve f) as described for DTT in the legend to Fig. 4. B, oxygen consumption of 3.5 mm TCEP in the presence of a mixture of 1 μm C30S/C33S plus 1 μm C130S/C133S, or 2 μm of the WT, C30S/C33S, or C130S/C133S, respectively. C, the relative activity of C30S/C33S plus C130S/C133S plotted against the incubation time. The activity of the WT Erv1 was set as 100%. D, oxygen consumption of 3.5 mm TCEP catalyzed by 1 μm C30S/C33S plus 1 μm C130S/C133S. The two mutants were preincubated for 5 min before TCEP was injected (curve a), TCEP was injected to C30S/C33S at 30 s followed by the addition of C130S/C133S at 300 s (curve b), and TCEP was injected to C130S/C133S at 30 s followed by the addition of C30S/C33S at 300 s (curve c).

Next, to confirm that Cys³⁰–Cys³³ can act as a shuttle disulfide, we performed the activity assay using TCEP as an electron donor in the presence of a total of 2 μm enzyme(s): WT Erv1, C30S/C33S, or C130S/C133S alone, or a mixture (preincubated for ∼5 min) of the two mutants (1 μm each), respectively. As show in Fig. 5B, although both mutants alone were inactive, the mixture of these two mutants showed a clear Erv1p oxidase activity. The rate of oxygen consumption was ∼0.36 μm s⁻¹, which was ∼24% that of the WT enzyme (1.5 μm s⁻¹) at the same condition (Fig. 5B). The result confirms that Cys³⁰–Cys³³ disulfide of a C130S/C133S mutant can activate C30S/C33S mutant via intersubunit or intermolecular electron transfer. If formation of a heterodimer between the two mutants is necessary for the activity, the activity will increase with the time of incubation. Interestingly, no obvious difference in the levels of activity was observed between 1, 5, and 20 min of incubation, but a slightly decreased activity was obtained when the proteins are preincubated for 2 h (Fig. 5C). Thus, these results suggest that heterodimer formation is not required. To confirm our finding, we carried out the experiments by incubating TCEP with one of the mutants first and followed by addition of the second mutants (Fig. 5D, curves b and c). As shown in Fig. 5D, the same rate of oxygen consumption as that of preincubated proteins (curve a) was obtained as soon as the second mutant was added in both cases. Taken together, we demonstrated experimentally for the first time that Cys³⁰–Cys³³ functionally acts as a shuttle disulfide passing electrons to the active site Cys¹³⁰–Cys¹³³ disulfide, and furthermore the electron transfer may occur between two different molecules via intermolecular interactions.

DISCUSSION

In this study, the structure and functional roles of all three individual disulfides of the yeast mitochondrial sulfhydryl oxidase Erv1p were analyzed using site-directed mutagenesis coupled with various biochemical and biophysical techniques. We show that whereas none of the three disulfides is essential for FAD-binding to Erv1p, they all play an important role in vitro. The specific roles played by each disulfide were deciphered systematically.

In this report, we provide direct experimental evidence to show that the N-terminal Cys³⁰–Cys³³ disulfide is required to shuttle electrons from the physiological substrate Mia40 to the active site Cys¹³⁰–Cys¹³³ disulfide. Although most in vitro studies on the function of FAD-dependent sulfhydryl oxidases have used a model substrate (e.g. DTT, reduced lysozyme, or E. coli thioredoxin), in this study, we used both artificial substrates (DTT and TCEP) and the physiological substrate, partially reduced Mia40, to address the functional mechanism of Erv1p. By comparison between the results, we are able to show that the active site disulfide Cys¹³⁰–Cys¹³³ is accessible to DTT but not to TCEP and Mia40; thus, in terms of biological relevance, TCEP is a better small molecule than DTT as substrate for Erv1p studies. C30S/C33S mutant was more active than the WT toward DTT oxidation but was enzymatically inactive toward Mia40c and TCEP oxidation. Together with the fact that TCEP is a stronger reductant and larger (250 Da) than DTT (154 Da), our results suggest that the active site disulfide is highly protected. It is not accessible to Mia40 and even to TCEP. The standard redox potential for Erv1p active site disulfide (Cys¹³⁰–Cys¹³³) has been determined to be of −150 mV (27). The redox potentials for the mitochondrial intermembrane space, GSH, and DTT were determined to be −255, −240, and −330 mV, respectively (31, 32), and an even lower value for TCEP was predicted (33). Accordingly, the active site disulfide of Erv1p would be thermodynamically unstable in the intermembrane space or against any of above thiol reducing reagents if it was not well protected. The inaccessible nature of the active site disulfide can also explain why DTT is a poor substrate for all members of Erv/ALR and Ero1 families in general. Our conclusion is also in agreement with the finding that GSH is not a good substrate for Erv1p (data not shown) and other Erv enzymes, because GSH is a weaker reductant than DTT and larger (307 Da) than TCEP. Thus, we provided direct experimental evidence for the current hypothesis that Cys³⁰–Cys³³ is required to serve as a shuttle disulfide transferring electrons from substrate to the active site Cys¹³⁰–Cys¹³³ disulfide.

More importantly, the Erv1p oxidase activity was partially recovered after mixing of the two individually inactive mutants, C30S/C33S and C130S/C133S (Fig. 5, B–D). An optimum of 50% of the WT Erv1p activity is expected for a perfect mixing and reassembled C30S/C33S-C130S/C133S Erv1. Because all known Erv/ALR enzymes form stable dimer or oligomers, the rate of dissociation is expected to be slow and probably in a time scale of min or even hours. Moreover, our results show that the enzyme activity was recovered rapid to ∼25% that of the WT Erv1p. Although we cannot exclude the possibility that the subunits rapidly scramble, our results suggest that this recovered activity was via intermolecular rather than intersubunit reaction. It seems likely that in the cells Cys³⁰–Cys³³ can shuttle electrons to Cys¹³⁰–Cys¹³³ through both intersubunit and intermolecular reactions and maybe even via intrachain shuttle. Similarly, for Ero1p a mechanism of both intra- and interchain electron transfer has been suggested (34).

Next, our study also suggests that Cys³⁰–Cys³³ disulfide and/or the flexible N-terminal region may also play a role in regulating the accessibility of the active site disulfide through a conformation change induced by interacting with Mia40 and thiol/disulfide exchange. Based on the CD measurements, a clear conformation change was observed for the N-terminal C30S/C33S mutant (data not shown). It suggests that a conformation change may occur in the flexible N-terminal region upon reduction of Cys³⁰–Cys³³ disulfide. Although the oxidase activity can be regained by mixing the active site C130S/C133S mutant with C30S/C33S, no enzyme activity was recovered by mixing C130S/C133S with Erv1-ΔN (a N-terminal 72-amino acid deleted Erv1) (data not shown). Thus, the interactions involving Erv1p N-terminal regions may play an important role during electron transfer from the shuttle disulfide to the active site disulfide and mediating the accessibility of the active site disulfide. In this way, the oxidase activity of Erv1p can be regulated effectively and specifically while circumventing or limiting nonspecific and harmful oxidation of protein thiols. Recently, a similar regulatory role played by a disulfide was demonstrated for both Ero1p and Ero1α (17–19). These regulatory disulfide bonds are among the nonconserved disulfides of Ero1 proteins. Although Erv1p has only three conserved disulfides, the location and the spacing between the two Cys residues of the shuttle disulfides (CX_nC, n = 1, 2, or 4) are not strictly conserved among the Erv/ALR enzymes. Accordingly, this disulfide may act as a regulative disulfide with substrate specificity.

Furthermore, this report provided direct evidence to show that CX₁₆C disulfide plays an important role in the overall stability of Erv1p. Based on our thermal denaturation and limited proteinase digestion studies, both disulfides (Cys¹³⁰–Cys¹³³ and Cys¹⁵⁹–Cys¹⁷⁶) of the C terminus contribute to the overall stability of the protein. Mutation of C130S/C133S decreases the T_m of Erv1p from 68 to 53 °C, and C159S/C176S has an apparent T_m of 38 °C. A fraction of C159S/C176S mutant was unfolded at temperature as low as 25 °C. Thus, this result provides a good explanation for the report that a single mutation of C159S results in a temperature-sensitive phenotype in yeast (35). Although the mutant yeast can grow at 24 °C, it cannot grow at 37 °C.

In summary, this study shows that all three pairs of disulfide bonds play an essential or important but different role in structure and function of Erv1p. Although the first CXXC motif (Cys³⁰–Cys³³) acts as shuttle disulfide and is involved in regulating the accessibility of the active site disulfide (Cys¹³⁰–Cys¹³³), the C-terminal CX₁₆C disulfide plays an important role in the overall stability of Erv1p. The well protected active site disulfide Cys¹³⁰–Cys¹³³ requires a shuttle disulfide to transfer electrons from substrate, and both intersubunit and intermolecular transfer can occur.