Quiescin sulfhydryl oxidase from Trypanosoma brucei: catalytic activity and mechanism of a QSOX family member with a single thioredoxin domain

Abstract

Quiescin sulfhydryl oxidase (QSOX) flavoenzymes catalyze the direct, facile, insertion of disulfide bonds into reduced unfolded proteins with the reduction of oxygen to hydrogen peroxide. To date, only QSOXs from vertebrates have been characterized enzymatically. These metazoan sulfhydryl oxidases have four recognizable domains: a redox-active thioredoxin (Trx) domain containing the first of three CxxC motifs (C(I)-C(II)), a second Trx domain with no obvious redox-active disulfide, a helix-rich domain, and then an Erv/ALR domain. This last domain contains the FAD moiety, a proximal C(III)-C(IV) disulfide, and a third CxxC of unknown function (C(V)-C(VI)). Plant and protist QSOXs lack the second Trx domain but otherwise appear to contain the same complement of redox centers. This work presents the first characterization of a single-Trx QSOX. Trypanosoma brucei QSOX was expressed in Escherichia coli using a synthetic gene and found to be a stable, monomeric, FAD-containing protein. Although evidently lacking an entire domain, TbQSOX shows catalytic activity and substrate specificity similar to the vertebrate QSOXs examined previously. Unfolded reduced proteins are more than 200-fold more effective substrates on a per thiol basis than glutathione and some 10-fold better than the parasite bisglutathione analogue, trypanothione. These data are consistent with a role for the protist QSOX in oxidative protein folding. Site-directed mutagenesis of each of the six cysteine residues (to serines) shows that the CxxC motif in the single-Trx domain is crucial for efficient catalysis of the oxidation of both reduced RNase and the model substrate dithiothreitol. As expected, the proximal disulfide C(III)-C(IV), which interacts with the flavin, is catalytically crucial. However, as observed with human QSOX1, the third CxxC motif shows no obvious catalytic role during the in vitro oxidation of reduced RNase or dithiothreitol. Pre-steady-state kinetics demonstrates that turnover in TbQSOX is limited by an internal redox step leading to 2-electron reduction of the FAD cofactor. In sum, the single-Trx domain QSOX studied here shows a striking similarity in enzymatic behavior to its double-Trx metazoan counterparts.

FIGURE 1

Domain organization of human and trypanosomal QSOX proteins and amino acid sequence of TbQSOX. Panels A and B show the domain organization of human and trypanosomal QSOX proteins respectively. CxxC motifs are shown as red bars; labeled as previously (10) and using the modified nomenclature implemented in this work. The flavin ring system of FAD is shown above the Erv/ALR label. Panel C represents the amino acid sequence (NCBI Acc. No. XP_845306), and predicted secondary structure of TbQSOX. The predicted signal sequence is underlined. Trx, HRR and Erv/ALR domains are highlighted in blue, orange and yellow respectively. A predicted transmembrane region is highlighted in pink. CxxC motifs are highlighted in red and underlined in boldface. The remaining cysteine residues are shown against a grey background.

FIGURE 2

UV/Vis spectrum of TbQSOX. The spectrum was recorded in 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA. The inset shows SDS-PAGE of TbQSOX under reducing (Red) and non-reducing (NR) conditions together with protein molecular weight standards in kDa.

FIGURE 3

pH dependence for the oxidation of DTT by TbQSOX. The ascending and descending limbs for the oxidation of 5 mM DTT were fit to pK values of 5.7 and 8.9, respectively, setting upper and lower limits of 2608 s⁻¹ and 0 s⁻¹.

FIGURE 4

Comparison of the turnover numbers of wild type TbQSOX and its CxxC mutants towards DTT and rRNase. Turnover numbers were measured at 25 °C in phosphate buffer, pH 7.5, using 5 mM DTT and 200 μM rRNase thiols (filled and open columns, respectively). Data were normalized with the wild-type protein set at 100 %.

FIGURE 5

Dithionite titrations of wild type, C^IS and HRR-Erv forms of TbQSOX. WT TbQSOX in 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA, was made anaerobic, and titrated at 25 °C with a standardized solution of sodium dithionite yielding curves 1–7. The inset plots the reduction of the oxidase as a function of electron equivalents added per flavin (■) together with comparable titrations with C^IS (○) and HRR-Erv (□) proteins.

FIGURE 6

Enzyme monitored turnover of DTT by TbQSOX. The oxidase was mixed with DTT to give final concentrations of 18.4 μM TbQSOX and 2.75 mM DTT in 50 mM phosphate buffer, pH 7.5, 25 °C containing 240 μM dissolved oxygen. Only selected spectra between 5 ms to 3 s are shown for clarity. The inset shows absorbance at 456 nm and 560 nm plotted as a function of time.

FIGURE 7

Anaerobic reduction of TbQSOX with DTT. Anaerobic solutions of TbQSOX and DTT (in 50 mM phosphate buffer, pH 7.5, containing 1 mM EDTA, 5 mM glucose, 5 nM glucose oxidase and 1 nM catalase) were mixed in a stopped-flow spectrophotometer at 25 °C to give final concentrations of 8 μM and 0.5 mM respectively. Panel A shows selected spectra from the diode-array detector and the inset follows the absorbance at 560 nm using a monochromator. Panel B plots observed rate constants as a function of the concentration of DTT. Appearance of the long wavelength species at 560 nm shows a limiting rate constant of 280 s⁻¹ with an apparent K_d of 0.66 mM (■). Disappearance of the long wavelength species (◆) and the reduction of flavin (○) show limiting rate constants of 18.8 s⁻¹ and 17.9 s⁻¹ respectively, both with an apparent K_d of 0.31 mM.

FIGURE 8

Simplified Model for Turnover in TbQSOX. Reduction of the C^I-C^II disulfide by DTT (k₁) yields form B. A thiolate to oxidized flavin charge-transfer species (represented by the curved dotted line: involving either, or both, of forms C and D) then forms with a limiting rate of 280 s⁻¹ (k₂). The charge-transfer absorbance decays (k₃) with a limiting rate of 18 s⁻¹ yielding reduced flavin (form E). The extent to which the interdomain redox reaction between forms C and D contribute to this rate liming step is unknown. The anticipated C4a adduct in the reduction of the flavin is omitted from the scheme because it has not been observed experimentally. The second substrate oxygen participates in k₄ by converting forms E to A. For simplicity, the scheme omits the possibility that DTT could reduce the C^I-C^II disulfide in forms D and E to generate 4-electron reduced TbQSOX prior to the oxidative half reaction (k₄). A contribution of 4-electron reduced oxidase to overall turnover would be favored by high DTT concentrations and low oxygen tensions.