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 CX(16)C 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 CX(16)C 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.

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.

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.

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.

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.

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).