Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Wang, Qi; Ang, Swee Kim; Ceh-Pavia, Efrain; Pang, Jiuxia; Lu, Hui

doi:10.1042/bsr20150144

Cited by 3 publications

(5 citation statements)

References 39 publications

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“…In practice, Mia40 is the physiological electron donor or upstream substrate of Erv1/ALR enzymes in mitochondria. To study the cytochrome c reductase kinetics of Erv1, reduced Mia40 was prepared as described previously . ScCytc at various concentrations was mixed with 160 μ m reduced Mia40 at 1 : 1 (v/v) ratio using stopped‐flow equipment under anaerobic conditions.…”

Section: Resultsmentioning

confidence: 99%

“…The WT and mutants of Erv1 proteins were expressed and purified as previously described . Briefly, the pET‐24a(+) plasmid containing the ERV1 gene was expressed in Escherichia coli Rosetta‐gami TM 2 cells (Sigma‐Aldrich Inc).…”

Section: Methodsmentioning

confidence: 99%

“…Essential for respiration and viability 1 enzymatic activity towards oxygen was measured using a Clark‐type oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK) in a 0.5 mL of reaction volume at 25 °C in BAE as previously described . When DTT was used as electron donor, bovine erythrocyte SOD1 (Sigma‐Aldrich Inc., cat no: 9054‐89‐1) was added at 10 unit·mL −1 to exclude the potential interference of superoxide ion .…”

Section: Methodsmentioning

confidence: 99%

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Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

et al. 2019

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Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html), a member of the flavin adenine dinucleotide‐dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped‐flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7‐ to 15‐fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic kcat and Km values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40‐Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space. Erv1 EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

et al. 2019

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“…For Erv1 (the WT and mutants), the pET‐24a(+) plasmid containing yeast ERV1 gene was expressed in Escherichia coli Rosetta‐gami™ 2 cells (Novagen, Merck KGaA, Darmstadt, Germany) and purified using Ni‐NTA (Ni 2+ ‐nitrilotriacetate) His‐bind beads (Novagen) as described previously . Further purification was done by size‐exclusion chromatography (SEC) using buffer AE (BAE: 50 m m Tris/HCl, 150 m m NaCl and 1 m m EDTA, pH 7.4) on a Superdex 200 or Superdex 200plus 100/300 GL column (GE Healthcare Bio‐sciences, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Pavia¹,

Tang²,

Liu

et al. 2019

The FEBS Journal

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Erv1, a member of the FAD-dependent Erv1/ALR disulphide bond generating enzyme family, is a key player of the MIA pathway. Although considerable progress has been made, the molecular mechanism of electron transfer within Erv1 is still not fully understood. The reduction potentials of the three redox centres were previously determined to be À320 mV for the shuttle disulphide, À150 mV for the active-site disulphide and À215 mV for FAD cofactor. However, it is unknown why FAD of Erv1 has such a low potential compared with other sulfhydryl oxidases, and why the shuttle disulphide has a potential as low as many of the stable structural disulphides of the substrates of MIA pathway. In this study, the three reduction potentials of Erv1 were reassessed using the wild-type and inactive mutants of Erv1 under anaerobic conditions. Our results show that the standard potentials for the shuttle and active-site disulphides are approximately À250 mV and À215~À260 mV, respectively, and the potential for FAD cofactor is À148 mV. Our results support a model that both disulphide bonds are redox-active, and electron flow in Erv1 is thermodynamically favourable. Furthermore, the redox behaviour of Erv1 was confirmed, for the first time using Mia40, the physiological electron donor of Erv1. Together with previous studies on proteins of MIA pathway, we conclude that electron flow in the MIA pathway is a thermodynamically favourable, smoothly downhill process for all steps. Database Erv1: EC 1.8.3.2.Abbreviations ALR, augmenter of liver regeneration; DTT, dithiothreitol; Erv, essential for respiration and viability; FAD, flavin adenine dinucleotide; IMS, intermembrane space; MIA, mitochondrial import and assembly; QSOX, quiescin sulfhydryl oxidase; TCEP, tris(2-carboxyethyl)phosphine.Electron flow mechanism of MIA40 pathway E. Ceh-Pavia et al.

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“…To evaluate the feasibility of the proposed IDA-SS for probing protein functions, the Erv1C served as a model protein. Erv1 is an essential component of the mitochondrial import and assembly pathway; , it has a cofactor of FAD. We purified His 6 -tagged yeast Erv1C (Holo form, with FAD) from E. coli cells as described previously .…”

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confidence: 99%

Surface-Enhanced Raman Scattering for Direct Protein Function Investigation: Controlled Immobilization and Orientation

Tang

Liu

et al. 2019

Anal. Chem.

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Surface-enhanced Raman spectroscopy (SERS) has exhibited great potential in protein identification and quantification. However, the poor spectral reproducibility, originating from random protein immobilization on SERS substrates, still makes it challenging for SERS to probe protein functions without any extrinsic Raman labels. Here, in our study, spacer molecules between proteins and SERS substrates are optimized for both biocompatible protein immobilization and Raman scattering enhancement. We have accordingly prepared iminodiacetic acid (IDA)-functionalized silver substrates, which are used for capturing His-tagged proteins via nickel−imidazole coordination. The controlled immobilization enables excellent SERS spectral reproducibility as evidenced by 6 polypeptides. Furthermore, the interactions between two model proteins, Erv1C (C-terminal domain of flavine adenine dinucleotide-dependent mitochondrial cytochrome c reductase Erv1) and AFP (alpha-fetoprotein), and their ligands Cyt c (cytochrome c) and ATRA (all-trans-retinoic acid) are examined, respectively. The results indicate that the IDA-functionalized silver substrates enable controlled protein immobilization and allow label-free protein function investigation by SERS. As a proof-of-concept study, the proposed functionalized SERS-active substrates combined with immobilized metal-affinity chromatography will be useful for mechanism studies on protein−ligand interactions, which is crucially important for understanding the structural basis of protein functional versatility and will contribute to the fields of drug design and biotechnology.

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Role of tryptophan residues of Erv1: Trp95 and Trp183 are important for its folding and oxidase function

Cited by 3 publications

References 39 publications

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Redox characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Surface-Enhanced Raman Scattering for Direct Protein Function Investigation: Controlled Immobilization and Orientation

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