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.
Highlights
28754 JOURNAL OF BIOLOGICAL CHEMISTRY import of a Cys-reduced substrate, Mia40 interacts with the substrate via intermolecular disulfide bond and shuttles a disulfide to its substrate
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]
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 wild type (WT)
Summary
Materials—4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid (AMS) and tris(2-carboxyethyl)phosphine (TCEP) were obtained from Molecular Probes (Invitrogen). 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]. Each bound AMS molecule increases the molecular mass of the protein ϳ0.5 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. Mass Spectrometry Analysis—The WT Erv (5 M) was incubated with 0 or 50 M freshly prepared Mia40c-pR for ϳ10 s, and the reaction was stopped by the addition of nonreducing SDS-PAGE sample buffer containing 4 mM iodoacetamide. The peptides were analyzed by mass spectrometry on a Bruker matrix-assisted laser desorption ionization time-offlight using a positive reflection method
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