Oxidative protein folding and the Quiescin-sulfhydryl oxidase family of flavoproteins

Abstract

Flavin-linked sulfhydryl oxidases participate in the net generation of disulfide bonds during oxidative protein folding in the endoplasmic reticulum. Members of the Quiescin-sulfhydryl oxidase (QSOX) family catalyze the facile direct introduction of disulfide bonds into unfolded reduced proteins with the reduction of molecular oxygen to generate hydrogen peroxide. Current progress in dissecting the mechanism of QSOX enzymes is reviewed, with emphasis on the CxxC motifs in the thioredoxin and Erv/ALR domains and the involvement of the flavin prosthetic group. The tissue distribution and intra- and extracellular location of QSOX enzymes are discussed, and suggestions for the physiological role of these enzymes are presented. The review compares the substrate specificity and catalytic efficiency of the QSOX enzymes with members of the Ero1 family of flavin-dependent sulfhydryl oxidases: enzymes believed to play key roles in disulfide generation in yeast and higher eukaryotes. Finally, limitations of our current understanding of disulfide generation in metazoans are identified and questions posed for the future.

FIG. 1.

Some reactions generating disulfide bonds. A number of transition metal ions, notably copper and iron, catalyze the oxidation of thiols to disulfides in aerobic solutions (A). Hydrogen peroxide is a slow, direct oxidant of free thiols (B). Quinones are used to oxidize thiols during catalysis by DsbB (C) and VKOR (D). Dehydroascorbate can accept reducing equivalents from protein thiols as shown in E. The flavoprotein sulfhydryl oxidases use the isoalloxazine ring to transfer reducing equivalents from reduced protein substrates to molecular oxygen (F).

FIG. 2.

The flavin ring of FAD and the proximal disulfide in Erv2p. The structure is taken from PDB 1JR8 (33). The C4a position of the isoalloxazine ring is indicated by a dark sphere and lies within van der Waals distance of the “charge-transfer” thiol of the proximal dithiol pair. The outer, or interchange, sulfur atom is indicated by a dark sphere.

FIG. 3.

Simplified reaction scheme for a generic sulfhydryl oxidase. A dithiol substrate is represented by a wavy line. The isoalloxazine ring of the bound flavin, and the adjacent redox-active disulfide, are included in the boxes. One of the cysteine residues forming this proximal disulfide interacts primarily with the flavin (the “charge-transfer” thiol). The second “interchange” cysteine (shown in boldface) forms mixed disulfides with external substrates, as in form 3.

FIG. 4.

Domain structure of one- and two-Trx QSOX enzymes. Metazoan QSOXs have two thioredoxin domains (labeled as Trx1 and Trx2 in A). Plants and protists lack all, or part, of the second thioredoxin domain (B). CxxC motifs are shown by the solid circles and identified in the text as C^I–C^II, C^III–C^IV, and C^V–C^VI. All QSOXs have a helix-rich region (HRR) fused to the flavin-binding domain (Erv/ALR).

FIG. 5.

Flow of reducing equivalents between the redox centers in QSOX. (A) shows the catalytic steps in the context of the domain structure of a metazoan QSOX. (B) depicts the interaction between thioredoxin and Erv/ALR domains using the crystal structures of the a domain of yeast PDI (95) and a subunit of Erv2p (33). The sulfur atoms participating in mixed disulfide bond formation (step 2) are shown as dark spheres.

FIG. 6.

Two models for oxidative folding catalyzed by sulfhydryl oxidases and PDI. QSOX oxidizes an unfolded protein directly and PDI isomerizes incorrectly paired disulfide bonds as they begin to accumulate (A). Oxidized PDI is the immediate oxidant for protein clients and is regenerated by Ero1-dependent reduction of molecular oxygen to form hydrogen peroxide (B). Reduced PDI is subsequently needed for the isomerization step.

FIG. 7.

Oxidative protein folding of riboflavin binding protein. Riboflavin binding to apo-RfBP is catalyzed by low nanomolar levels of QSOX and high micromolar concentrations of reduced PDI.

FIG. 8.

PDI and Ero1 catalyze hydrogen peroxide formation at the expense of reduced glutathione. This system replaces reduced unfolded client proteins (Fig. 6B) with reduced glutathione in the ER lumen. These reactions could lead to unwanted loss of reducing equivalents as hydrogen peroxide (see text).

FIG. 9.

EST frequencies for four sulfhydryl oxidases in human tissues. The data, from Unigene Build 221, are expressed as frequencies per million ESTs. The column heights represent the aggregate of QSOX1 and QSOX2 EST frequencies, with the open and filled components indicating the ratio of QSOX1 to QSOX2 frequencies, respectively. The solid and dotted lines represent the corresponding EST frequencies for Ero1L and Ero1LB. QSOX1 is particularly abundant in lung tissue (broken vertical axis). For comparison, a chondrosarcoma cell line shows a frequency of 6746 QSOX1 ESTs per million.