The N‐terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for <i>in vivo</i> activity and interacts with the primary redox centre

Hofhaus, Götz; Lee, Jeung-Eun; Tews, Ivo; Rosenberg, Beate; Lisowsky, Thomas

doi:10.1046/j.1432-1033.2003.03519.x

Cited by 75 publications

(120 citation statements)

References 27 publications

(49 reference statements)

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“…While a crystal structure of reduced Erv2p has yet to appear, the rigidity of this small 4-helix bundle make this possibility appear unlikely. Furthermore, mutation of CYS II to either SER or ALA in ERV1 (46) and in ALR (Farrell & Thorpe unpublished observation) shows the now-isolated CYS I can indeed form prominent chargetransfer absorbance at wavelengths > 530 nm. Another possible explanation is that the pK values of CYS I in forms B or C in Scheme 2 are too high to observe significant thiolate to flavin charge-transfer at pH 7.5.…”

Section: The Erv/alr Family Of Sulfhydryl Oxidases: a Comparisonmentioning

confidence: 99%

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

2007

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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...

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Section: The Erv/alr Family Of Sulfhydryl Oxidases: a Comparisonmentioning

confidence: 99%

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

2007

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“…The equivalent residue is Arg in Erv-like domains of human and rat augmenter-of-liver-regeneration proteins (35,36). Furthermore, some flavoenzymes that contain an ERV͞ALR domain exhibit prominent thiolate-to-flavin CT bands (37,38). It is of prime interest to ask whether Erv2p͞Ero1 undergoes any spectroscopic transition in the course of its catalytic reactions.…”

Section: Do Quinone-and Flavin-dependent Disulfide Oxidoreductases Shmentioning

confidence: 99%

Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB

Inaba

Takahashi

Ito

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Recent studies have revealed numerous examples in which oxidation and reduction of cysteines in proteins are integrated into specific cascades of biological regulatory systems. In general, these reactions proceed as thiol-disulfide exchange events. However, it is not exactly understood how a disulfide bond is created de novo. DsbB, an Escherichia coli plasma membrane protein, is one of the enzymes that create a new disulfide bond within itself and in DsbA, the direct catalyst of protein disulfide bond formation in the periplasmic space. DsbB is associated with a cofactor, either ubiquinone or menaquinone, as a source of an oxidizing equivalent. The DsbB-bound quinone undergoes transition to a pink ( max, Ϸ500 nm, ubiquinone) or violet ( max, Ϸ550 nm, menaquinone)-colored state during the course of the DsbB enzymatic reaction. Here we show that not only the thiolate form of Cys-44 previously suggested but also Arg-48 in the ␣-helical arrangement is essential for the quinone transition. Quantum chemical simulations indicate that proper positioning of thiolate anion and ubiquinone in conjunction with positively charged guanidinium moiety of arginine allows the formation of a thiolate-ubiquinone charge transfer complex with absorption peaks at Ϸ500 nm as well as a cysteinylquinone covalent adduct. We propose that the charge transfer state leads to the transition state adduct that accepts a nucleophilic attack from another cysteine to generate a disulfide bond de novo. A similar mechanism is conceivable for a class of eukaryotic dithiol oxidases having a FAD cofactor.FAD ͉ ubiquinone ͉ DsbA

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“…S1). Previous genetic studies in S. cerevisiae demonstrated that all Cys residues of Erv1 were required for its function in vivo (12). These motifs at the N-or C-terminus have been proposed to work as electron shuttling from the substrate to the FAD molecule.…”

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confidence: 99%

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

Banci

Bertini

Calderone

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

115

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Oxidative protein folding in the mitochondrial intermembrane space requires the transfer of a disulfide bond from MIA40 to the substrate. During this process MIA40 is reduced and regenerated to a functional state through the interaction with the flavin-dependent sulfhydryl oxidase ALR. Here we present the mechanistic basis of ALR-MIA40 interaction at atomic resolution by biochemical and structural analyses of the mitochondrial ALR isoform and its covalent mixed disulfide intermediate with MIA40. This ALR isoform contains a folded FAD-binding domain at the C-terminus and an unstructured, flexible N-terminal domain, weakly and transiently interacting one with the other. A specific region of the N-terminal domain guides the interaction with the MIA40 substrate binding cleft (mimicking the interaction of the substrate itself), without being involved in the import of ALR. The hydrophobicity-driven binding of this region ensures precise protein-protein recognition needed for an efficient electron transfer process.

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The N‐terminal cysteine pair of yeast sulfhydryl oxidase Erv1p is essential for in vivo activity and interacts with the primary redox centre

Cited by 75 publications

References 27 publications

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

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

Critical role of a thiolate-quinone charge transfer complex and its adduct form in de novo disulfide bond generation by DsbB

Molecular recognition and substrate mimicry drive the electron-transfer process between MIA40 and ALR

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