Going through the barrier: coupled disulfide exchange reactions promote efficient catalysis in quiescin sulfhydryl oxidase

Abstract

The quiescin sulfhydryl oxidase (QSOX) family of enzymes generates disulfide bonds in peptides and proteins with the reduction of oxygen to hydrogen peroxide. Determination of the potentials of the redox centers in Trypanosoma brucei QSOX provides a context for understanding catalysis by this facile oxidant of protein thiols. The CXXC motif of the thioredoxin domain is comparatively oxidizing (E'0 of -144 mV), consistent with an ability to transfer disulfide bonds to a broad range of thiol substrates. In contrast, the proximal CXXC disulfide in the ERV (essential for respiration and vegetative growth) domain of TbQSOX is strongly reducing (E'0 of -273 mV), representing a major apparent thermodynamic barrier to overall catalysis. Reduction of the oxidizing FAD cofactor (E'0 of -153 mV) is followed by the strongly favorable reduction of molecular oxygen. The role of a mixed disulfide intermediate between thioredoxin and ERV domains was highlighted by rapid reaction studies in which the wild-type CGAC motif in the thioredoxin domain of TbQSOX was replaced by the more oxidizing CPHC or more reducing CGPC sequence. Mixed disulfide bond formation is accompanied by the generation of a charge transfer complex with the flavin cofactor. This provides thermodynamic coupling among the three redox centers of QSOX and avoids the strongly uphill mismatch between the formal potentials of the thioredoxin and ERV disulfides. This work identifies intriguing mechanistic parallels between the eukaryotic QSOX enzymes and the DsbA/B system catalyzing disulfide bond generation in the bacterial periplasm and suggests that the strategy of linked disulfide exchanges may be exploited in other catalysts of oxidative protein folding.

Keywords: Disulfide; Enzyme Mechanisms; Oxidase; Oxidation-Reduction; Protein Folding; QSOX; Quiescin Sulfhydryl Oxidase; Redox; Sulfhydryl.

FIGURE 1.

Domain organization and crystal structures of TbQSOX. A represents the domain structure of TbQSOX: the three CXXC motifs (C^IXXC^II, C^IIIXXC^IV, and C^VXXC^VI) together with the TRX domain, HRR, and ERV domain shown in blue, gray, and green, respectively. The flow of reducing equivalents from reduced protein substrate to molecular oxygen is schematically depicted with arrows 1–4. The third conserved CXXC disulfide (C^VXXC^VI) is not required for the oxidation of small molecule or protein substrates in vitro and is not considered further here. B shows the crystal structure of an open form of the enzyme (Protein Data Bank code 3QCP) using the same domain coloring and orientation as in A. The CXXC sulfur atoms are depicted as yellow spheres, and the FAD is depicted using yellow sticks. Cysteine to alanine mutations at Cys^II and Cys^IV allowed the oxidative capture of the interdomain Cys^I-Cys^III mixed disulfide shown in the closed form of TbQSOX in C (Protein Data Bank code 3QD9). The dashed lines in B and C depict a mobile loop not resolved in the crystal structures.

FIGURE 2.

Redox potential determination of the thioredoxin domain CXXC motif in TbQSOX. A, SDS-PAGE showing the redox equilibration of TbQSOX-TRX incubated with glutathione redox buffers where the proportion of reduced glutathione increases from left to right. The portion of protein containing reduced CXXC reacts with MM(PEG)₂₄ and thus runs as a higher molecular weight band than the oxidized protein. Minor intermediate bands corresponding to protein containing a mixed disulfide species with glutathione (*) were excluded from analysis. A portion of the protein becomes over-reduced at high GSH concentrations. B, gel shift controls using 20 mm GSSG or 20 mm GSH in the presence or absence of MM(PEG)₂₄. High levels of reduced GSH cause the majority of the protein to become over-reduced. C, the band intensities of reduced forms and oxidized protein in A were quantified by densitometry, and the fractions of reduced protein from four independent experiments were plotted, and a non-linear fit of the data yielded a redox potential of −144 ± 1 mV (see “Experimental Procedures”). Error bars represent S.E.

FIGURE 3.

Determination of the redox potential of the proximal CXXC disulfide in TbQSOX. 5-Deaza-FAD-substituted TbQSOX was prepared as described under “Experimental Procedures.” A, the spectrum of the protein was recorded before and after reduction with 5 mm DTT (red and blue lines, respectively). The ∼5-nm blue shift upon reduction of the proximal disulfide resulted in maximum differences in absorbance between oxidized and reduced forms centered around 445 nm (inset), which was used to calculate the fraction of reduced enzyme with increasing ratios of reduced to oxidized DTT (see text). B, the fractions of reduced enzyme were plotted, and a non-linear fit to the data from three independent experiments gave a redox potential of −273 ± 3 mV for the proximal disulfide. Error bars represent S.E.

FIGURE 4.

The redox potential of the FAD cofactor in TbQSOX. Full-length TbQSOX was equilibrated with reduced and oxidized glutathione under rigorously anaerobic conditions. A, reduction of the FAD was quantified by the decline in absorbance at 456 nm with increasing GSH additions. B, a non-linear fit of the fraction of reduced FAD yields a redox potential of −153 ± 1 mV. Error bars represent S.E.

FIGURE 5.

Turnover number of the C^IPHC^II mutation of TbQSOX with DTT. The main panel represents turnover numbers up to 1 mm DTT. The inset shows the linear dependence of the turnover number of the mutant enzyme at concentrations above 1 mm DTT. Error bars represent S.E.

FIGURE 6.

Steady state spectra of wild-type, C^IPHC^II, and C^IGPC^II mutations of TbQSOX during the oxidation of DTT in air-saturated buffer. A, wild-type TbQSOX shows a small amount of charge transfer absorbance in the steady state observed when the enzyme is mixed aerobically with 5 mm DTT in 50 mm phosphate buffer, pH 7.5, 25 °C. By contrast, the C^IPHC^II mutant (red) lacks any detectable charge transfer species, and the C^IGPC^II mutant (blue) shows a strong charge transfer absorbance. B–D, the corresponding time courses for the absorbance changes at 456 and 580 nm, respectively, are shown.

FIGURE 7.

Reduction of the C^IGPC^II mutant under anaerobic conditions and analysis of the dependence of the apparent rate constants for formation and decay of the charge transfer complex on DTT concentration. A, anaerobic stopped-flow spectra demonstrating the formation of a long-wavelength charge transfer intermediate and subsequent disappearance of this species as flavin is reduced. For clarity, only select spectra are shown (starting with red, dark red, and brown traces). B, apparent rate constants for the formation (solid squares) and decay (gray diamonds) of the long-wavelength charge transfer species characterized at 580 nm.

FIGURE 8.

Schematic of free energy coordinates and equilibria depicting overall catalysis by TbQSOX and DsbA/B. A shows a schematic free energy coordinate for TbQSOX. The transfer of electrons from reduced substrate to C^IXXC^II of TbQSOX is energetically favorable, whereas the subsequent intramolecular transfer of electrons to C^IIIXXC^IV is strongly unfavorable based on free energy coordinates alone. The reduction of the FAD prosthetic group and the final transfer from dihydroflavin to molecular oxygen (73) are both favored energetically. A also depicts the interconversion of selected 2-electron reduced forms in TbQSOX. B presents the corresponding schemes for DsbA/B. The redox potentials for DsbB disulfides in B are the average of those of Regeimbal and Bardwell (74) and Inaba and Ito (61). Cysteine residues are labeled by their position in the sequence of DsbA and DsbB.