Erv2p:  Characterization of the Redox Behavior of a Yeast Sulfhydryl Oxidase

Abstract

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4-electrons with a mid-point potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn 2+ , full reduction of Erv2p requires 6-electrons. Zn 2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.Over the last decade a number of eukaryotic flavin-dependent sulfhydryl oxidases have been described that catalyze the net generation of disulfide bonds:They include: yeast ERV1 (essential for respiration and vegetative growth) (1) and its mammalian counterpart augmenter of liver regeneration, ALR, (2-4); yeast Erv2p (5-7); an Arabidopsis homolog of these proteins (8,9); yeast and its vertebrate orthologs Ero1α and Ero1β (15,16); and finally larger multidomain sulfhydryl oxidases, containing an ERV/ALR module fused to a redox active thioredoxin domain, that are found principally in multicellular organisms (17)(18)(19)(20) The ERV/ALR proteins and their larger cousins in the QSOX family all contain a diminutive helix-rich flavin binding domain first reported for yeast Erv2p by Fass and coworkers (6) and for rat ALR by Rose and colleagues (4). Subunit A of the Erv2p homodimer is shown in the foreground of Figure 1. The isoalloxazine ring is inserted at the end of a bundle of four helices in the A subunit with its C2/N3 region exposed to solvent (6). The proximal disulfide C121-124 is placed so that the sulfur of C124 is adjacent (3.4 Å) to the C 4a position of the flavin. This locus is consistent with i...

Zinc Can Play Chaperone-like and Inhibitor Roles during Import of Mitochondrial Small Tim Proteins

“…Consistent with our results, a similar inhibitory effect of zinc on the yeast sulfhydryl oxidase Erv2 was reported previously (22), suggesting that zinc may be an inhibitor for the activity of sulfhydryl oxidases in general. Furthermore, our results are consistent with the findings that different cell compartments are biased toward different elements (2, 28 -30), and whereas the cytosol has a high level of zinc storage, the zinc level in mitochondria is low and regulated independently (31).…”

“…Reduced proteins were always prepared fresh immediately before use typically by incubation of oxidized proteins with 1-2 mM TCEP for 60 -90 min at room temperature, followed by gel filtration using a Superdex 75 column to remove TCEP. Erv1-His 6 was expressed in Escherichia coli Rosetta-gami TM 2 (Novagen) and purified using His-tagged affinity beads as described for Erv2 (22). Then, Erv1 was dialyzed against buffer A and supplemented with 50 M FAD before being stored at Ϫ80°C.…”

Human Quiescin-Sulfhydryl Oxidase, QSOX1: Probing Internal Redox Steps by Mutagenesis

“…When cysteines in the CxxC motif of the HsQSOX Trx1 domain were mutated to serine (C70S and C73S), residual activities of about 7.5% of wild-type with DTT and 1.5% with reduced RNase were observed ( DTT and, correspondingly,10,17,27,43).…”

“…To address the role of each CxxC motif in HsQSOX1, we made single Cys-to-Ala or -Ser mutants for all six cysteine residues; for one pair (509/512) we also generated an SxxS double-mutant (see Methods). Activities of mutant proteins were initially measured using 5 mM DTT or 38 μM reduced RNase (0.3 mM thiols) as substrates (Table 3).When cysteines in the CxxC motif of the HsQSOX Trx1 domain were mutated to serine (C70S and C73S), residual activities of about 7.5% of wild-type with DTT and 1.5% with reduced RNase were observed ( DTT and, correspondingly,10,17,27,43).We next mutated the two cysteine residues that comprise the proximal disulfide C449-452 in HsQSOX1 (corresponding to C121-124 in Erv2p; Figure 1). In Erv2p, C121, the residue furthest from the isoalloxazine ring, would correspond to the interchange thiol (following the nomenclature developed for the pyridine nucleotide disulfide oxidoreductases (22, 23)) forming mixed disulfides with attacking thiolate nucleophiles.…”

Adenoviral Gene Transfer of Hepatic Stimulator Substance Confers Resistance Against Hepatic Ischemia–Reperfusion Injury by Improving Mitochondrial Function

“…HSS exists in either a longer (with 205 amino acids) or a shorter form (with only 125 amino acids). In addition, together with Erv1p, a Saccharomyces cerevisiae homolog, HSS is a member of the new ALR/ERV1 protein family, which belongs to the sulfhydryl oxidase (SOX) enzymes that participate in disulfide bond formation (Wang et al, 2007;Daithankar et al, 2012). Structurally, HSS contains the conserved CXXC motif of SOX and a noncovalent flavin adenine dinucleotide (FAD) adjacent to CXXC, and these domains are vital to its catalytic activity (Wu et al, 2003).…”