Human quiescin-sulfhydryl oxidase, QSOX1: probing internal redox steps by mutagenesis

Abstract

The flavoprotein quiescin-sulfhydryl oxidase (QSOX) rapidly inserts disulfide bonds into unfolded, reduced proteins with the concomitant reduction of oxygen to hydrogen peroxide. This study reports the first heterologous expression and enzymological characterization of a human QSOX1 isoform. Like QSOX isolated from avian egg white, recombinant HsQSOX1 is highly active toward reduced ribonuclease A (RNase) and dithiothreitol but shows a >100-fold lower k cat/ K m for reduced glutathione. Previous studies on avian QSOX led to a model in which reducing equivalents were proposed to relay through the enzyme from the first thioredoxin domain (C70-C73) to a distal disulfide (C509-C512), then across the dimer interface to the FAD-proximal disulfide (C449-C452), and finally to the FAD. The present work shows that, unlike the native avian enzyme, HsQSOX1 is monomeric. The recombinant expression system enabled construction of the first cysteine mutants for mechanistic dissection of this enzyme family. Activity assays with mutant HsQSOX1 indicated that the conserved distal C509-C512 disulfide is dispensable for the oxidation of reduced RNase or dithiothreitol. The four other cysteine residues chosen for mutagenesis, C70, C73, C449, and C452, are all crucial for efficient oxidation of reduced RNase. C452, of the proximal disulfide, is shown to be the charge-transfer donor to the flavin ring of QSOX, and its partner, C449, is expected to be the interchange thiol, forming a mixed disulfide with C70 in the thioredoxin domain. These data demonstrate that all the internal redox steps occur within the same polypeptide chain of mammalian QSOX and commence with a direct interaction between the reduced thioredoxin domain and the proximal disulfide of the Erv/ALR domain.

FIGURE 1

Domain organization of a metazoan QSOX and sequence comparisons with the “a” domain of yeast Pdi1p and yeast Erv2p. Partial sequences of human (HsQSOX1) and avian (GgQSOX1) enzymes are compared with an alignment to the “a” domain of yeast Pdi1p (ScPDI1a; NCBI Accession NP_009887.1) and the Erv/ALR domain of yeast Erv2p (ScErv2; NCBI Accession Q12284). The recognized domains of the short form of metazoan QSOX (Trx1, Trx2, HRR, and Erv/ALR) are boxed. Presumed redox active disulfides are shown in bold reverse type; the remaining six cysteines are underlined. Residues in human and avian QSOX1 that are conserved with the PDI1a or ScErv2 sequences are shown by the asterisks.

FIGURE 2

Crystal structure and the proposed flow of reducing equivalents in the yeast Erv2p homodimer. The proposed flow of reducing equivalents from the reductant (Pdi1p) to molecular oxygen is depicted from left to right using a series of arrows. An equivalent set of redox centers are shown unlabelled at the back of the homodimer. C150–167 is a structural disulfide in Erv2p that is absent in the multi-domain Quiescin-sulfhydryl oxidase (QSOX).

FIGURE 3

Suggested flow of reducing equivalents during oxidation of a reduced protein by avian QSOX1. The client reduced protein is oxidized by the Trx1 domain of QSOX followed by transfer of the pair of reducing equivalents to the Erv/ALR domain. The model incorporates an intersubunit disulfide exchange between distal and proximal CxxC motifs of a dimeric Erv/ALR domain as shown in Figure 2 (see Text).

FIGURE 4

UV-visible spectrum of recombinant human QSOX1. The spectrum of 2.9 μM enzyme was recorded in 50 mM phosphate pH 7.5, containing 1 mM EDTA and was corrected for small amounts of scattering material as described in the Experimental Procedures. The top spectrum is multiplied by 5 to show details of the flavin envelope.

FIGURE 5

UV-Visible spectra of cysteine mutants of the proximal disulfide of HsQSOX1. Spectra of the following mutants of HsQSOX1 in phosphate buffer pH 7.5: C449A (solid line), C449S (short dashed line), and C452S (long dashed line). Spectra were normalized to correspond to a concentration of 3.2 μM using the extinction coefficients reported in the Experimental Procedures.

FIGURE 6

Proposed mechanism for the transfer of reducing equivalents between flavin and the reduced proximal disulfide in QSOX. The interchange and charge transfer thiols correspond to C449 and C452 respectively.

FIGURE 7

Revised model for flow of reducing equivalents in QSOX. This model involves a transitory mixed disulfide between C70 of Trx1 and C452 of the proximal disulfide in the Erv/ALR domain (see the Text). The distal CxxC disulfide (C509–512) would be on the opposite face of the Erv/ALR domain more than 25 Å from C452.