Publications

• A flavin-dependent sulfhydryl oxidase in bovine milk.

  Jaje J., Wolcott H.N., Fadugba O., Cripps D., Yang A.J., Mather I.H., Thorpe C.

  Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H ... >> More

  Both metal and flavin-dependent sulfhydryl oxidases catalyze the net generation of disulfide bonds with the reduction of oxygen to hydrogen peroxide. The first mammalian sulfhydryl oxidase to be described was an iron-dependent enzyme isolated from bovine milk whey (Janolino, V.G., and Swaisgood, H.E. (1975) J. Biol. Chem. 250, 2532-2537). This protein was reported to contain 0.5 atoms of iron per 89 kDa subunit and to be completely inhibited by ethylenediaminetetraacetate (EDTA). However the present work shows that a soluble 62 kDa FAD-linked and EDTA-insensitive sulfhydryl oxidase apparently constitutes the dominant disulfide bond-generating activity in skim milk. Unlike the metalloenzyme, the flavoprotein is not associated tightly with skim milk membranes. Sequencing of the purified bovine enzyme (>70% coverage) showed it to be a member of the Quiescin-sulfhydryl oxidase (QSOX) family. Consistent with its solubility, this bovine QSOX1 paralogue lacks the C-terminal transmembrane span of the long form of these proteins. Bovine milk QSOX1 is highly active toward reduced RNase and with the model substrate dithiothreitol. The significance of these new findings is discussed in relation to the earlier reports of metal-dependent sulfhydryl oxidases. << Less

  Biochemistry 46:13031-13040(2007) [PubMed] [EuropePMC]

• Thioxidase, a new sulfhydryl-oxidizing enzyme from Piricularia oryzae and Polyporus versicolor.

  NEUFELD H.A., GREEN L.F., LATTERELL F.M., WEINTRAUB R.L.

  J Biol Chem 232:1093-1099(1958) [PubMed] [EuropePMC]

• Disulfide formation in the ER and mitochondria: two solutions to a common process.

  Riemer J., Bulleid N., Herrmann J.M.

  The endoplasmic reticulum (ER) was long considered to be the only compartment of the eukaryotic cell in which protein folding is accompanied by enzyme-catalyzed disulfide bond formation. However, it has recently become evident that cells harbor a second oxidizing compartment, the mitochondrial int ... >> More

  The endoplasmic reticulum (ER) was long considered to be the only compartment of the eukaryotic cell in which protein folding is accompanied by enzyme-catalyzed disulfide bond formation. However, it has recently become evident that cells harbor a second oxidizing compartment, the mitochondrial intermembrane space, where disulfide formation facilitates protein translocation from the cytosol. Moreover, protein oxidation has been implicated in many mitochondria-associated processes central for human health such as apoptosis, aging, and regulation of the respiratory chain. Whereas the machineries of ER and mitochondria both form disulfides between cysteine residues, they do not share evolutionary origins and exhibit distinct mechanistic properties. Here, we summarize the current knowledge of these oxidation systems and discuss their functional similarities and differences. << Less

  Science 324:1284-1287(2009) [PubMed] [EuropePMC]

• Properties of a flavoprotein sulfhydryl oxidase from rat seminal vesicle secretion.

  Ostrowski M.C., Kistler W.S.

  Rat seminal vesicle secretion is a rich source of a flavoprotein oxidase that acts upon sulfhydryl compounds. The enzyme was obtained in homogeneous form as previously described [Ostrowski, M. C., Kistler, W. S., & Williams-Ashman, H. G. (1979) Biochem. Biophy. Res. Commun. 87, 171-176] and charac ... >> More

  Rat seminal vesicle secretion is a rich source of a flavoprotein oxidase that acts upon sulfhydryl compounds. The enzyme was obtained in homogeneous form as previously described [Ostrowski, M. C., Kistler, W. S., & Williams-Ashman, H. G. (1979) Biochem. Biophy. Res. Commun. 87, 171-176] and characterized with respect to prosthetic group, size, reaction stoichiometry, and substrate specificity. On the basis of its behavior during zone sedimentation, gel filtration, and electrophoresis in the presence of sodium dodecyl sulfate, it appears to be a monomeric enzyme of about 66 000 daltons. Acid denaturation liberates 1 mol of flavin adenine dinucleotide (FAD) per mol of enzyme. The reaction catalyzed was shown to be 2RSH + O2 leads to H2O2. Superoxide formation could be demonstrated. Unlike many flavoprotein oxidases, the enzyme failed to form a bleached complex with sulfite. The enzyme accepts a variety of small sulfhydryl compounds as substrates, including glutathione, cysteine, dithiothreitol, and 2-mercaptoethanol. Michaelis-Menten kinetics were obtained with these substrates providing disulfide contamination was initially eliminated by treating thiols with borohydride. The KM for glutathione was 4.4 mM with a Vmax estimated as 660 mumol per min per mg of protein. The enzyme was capable of markedly enhancing the rate of renaturation of fully reduced ribonuclease. The physiological function of the enzyme is not yet clear, though several possibilities are discussed. << Less

  Biochemistry 19:2639-2645(1980) [PubMed] [EuropePMC]

• A flavoprotein oxidase defines a new endoplasmic reticulum pathway for biosynthetic disulphide bond formation.

  Sevier C.S., Cuozzo J.W., Vala A., Aaslund F., Kaiser C.A.

  Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bo ... >> More

  Ero1 and Pdi1 are essential elements of the pathway for the formation of disulphide bonds within the endoplasmic reticulum (ER). By screening for alternative oxidation pathways in Saccharomyces cerevisiae, we identified ERV2 as a gene that when overexpressed can restore viability and disulphide bond formation to an ero1-1 mutant strain. ERV2 encodes a luminal ER protein of relative molecular mass 22,000. Purified recombinant Erv2p is a flavoenzyme that can catalyse O2-dependent formation of disulphide bonds. Erv2p transfers oxidizing equivalents to Pdi1p by a dithiol-disulphide exchange reaction, indicating that the Erv2p-dependent pathway for disulphide bond formation closely parallels that of the previously identified Ero1p-dependent pathway. << Less

  Nat. Cell Biol. 3:874-882(2001) [PubMed] [EuropePMC]

• Generating disulfides enzymatically: reaction products and electron acceptors of the endoplasmic reticulum thiol oxidase Ero1p.

  Gross E., Sevier C.S., Heldman N., Vitu E., Bentzur M., Kaiser C.A., Thorpe C., Fass D.

  Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and th ... >> More

  Ero1p is a key enzyme in the disulfide bond formation pathway in eukaryotic cells in both aerobic and anaerobic environments. It was previously demonstrated that Ero1p can transfer electrons from thiol substrates to molecular oxygen. However, the fate of electrons under anaerobic conditions and the final fate of electrons under aerobic conditions remained obscure. To address these fundamental issues in the Ero1p mechanism, we studied the transfer of electrons from recombinant yeast Ero1p to various electron acceptors. Under aerobic conditions, reduction of molecular oxygen by Ero1p yielded stoichiometric hydrogen peroxide. Remarkably, we found that reduced Ero1p can transfer electrons to a variety of small and macromolecular electron acceptors in addition to molecular oxygen. In particular, Ero1p can catalyze reduction of exogenous FAD in solution. Free FAD is not required for the catalysis of dithiol oxidation by Ero1p, but it is sufficient to drive disulfide bond formation under anaerobic conditions. These findings provide insight into mechanisms for regenerating oxidized Ero1p and maintaining disulfide bond formation under anaerobic conditions in the endoplasmic reticulum. << Less

  Proc Natl Acad Sci U S A 103:299-304(2006) [PubMed] [EuropePMC]

• The multiple functions of thiooxidase.

  AURBACH G.D., JAKOBY W.B.

  J Biol Chem 237:565-568(1962) [PubMed] [EuropePMC]

• A role for cytochrome c and cytochrome c peroxidase in electron shuttling from Erv1.

  Dabir D.V., Leverich E.P., Kim S.K., Tsai F.D., Hirasawa M., Knaff D.B., Koehler C.M.

  Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all red ... >> More

  Erv1 is a flavin-dependent sulfhydryl oxidase in the mitochondrial intermembrane space (IMS) that functions in the import of cysteine-rich proteins. Redox titrations of recombinant Erv1 showed that it contains three distinct couples with midpoint potentials of -320, -215, and -150 mV. Like all redox-active enzymes, Erv1 requires one or more electron acceptors. We have generated strains with erv1 conditional alleles and employed biochemical and genetic strategies to facilitate identifying redox pathways involving Erv1. Here, we report that Erv1 forms a 1:1 complex with cytochrome c and a reduced Erv1 can transfer electrons directly to the ferric form of the cytochrome. Erv1 also utilized molecular oxygen as an electron acceptor to generate hydrogen peroxide, which is subsequently reduced to water by cytochrome c peroxidase (Ccp1). Oxidized Ccp1 was in turn reduced by the Erv1-reduced cytochrome c. By coupling these pathways, cytochrome c and Ccp1 function efficiently as Erv1-dependent electron acceptors. Thus, we propose that Erv1 utilizes diverse pathways for electron shuttling in the IMS. << Less

  EMBO J 26:4801-4811(2007) [PubMed] [EuropePMC]

• Augmenter of liver regeneration: a flavin-dependent sulfhydryl oxidase with cytochrome c reductase activity.

  Farrell S.R., Thorpe C.

  Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a rece ... >> More

  Augmenter of liver regeneration (ALR; hepatopoietin) is a recently discovered enigmatic flavin-linked sulfhydryl oxidase. An N-terminal His-tagged construct of the short form of the human protein has been overexpressed in Escherichia coli. Several lines of evidence suggest that, contrary to a recent report, human ALR is a disulfide-bridged dimer (linked via C15-C124) with two free cysteine residues (C74 and 85) per monomer. The C15-124 disulfides are not critical for dimer formation and have insignificant impact on the dithiothreitol (DTT) oxidase activity of ALR. Although the crystal structure of rat ALR shows a proximal disulfide (C62-C65) poised to interact with the FAD prosthetic group [Wu, C. K., Dailey, T. A., Dailey, H. A., Wang, B. C., and Rose, J. P. (2003) Protein Sci. 12, 1109-1118], only flavin reduction is evident during redox titrations of the enzyme. ALR forms large amounts of neutral semiquinone during aerobic turnover with DTT. This semiquinone arises, in part, by comproportionation between flavin centers within the dimer. Surprisingly, cytochrome c is about a 100-fold better electron acceptor for ALR than oxygen when DTT is the reducing substrate. These data suggest that this poorly understood flavoenzyme may not function as a sulfhydryl oxidase within the mitochondrial intermembrane space but may communicate with the respiratory chain via the mediation of cytochrome c. << Less

  Biochemistry 44:1532-1541(2005) [PubMed] [EuropePMC]

• Aspergillus niger sulfhydryl oxidase.

  de la Motte R.S., Wagner F.W.

  A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a sub ... >> More

  A procedure for the isolation of a sulfhydryl oxidase from an Aspergillus niger cell suspension involved three major steps and yielded enzyme preparations exhibiting a single but diffuse protein-containing zone when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a subunit molecular weight estimated to be 53,000. Sedimentation equilibrium experiments indicated a native molecular weight of 106,000. Analyses for sugar residues showed that the enzyme is a glycoprotein, containing 20.3% neutral hexose and 1.9% aminohexose by weight. This enzyme catalyzed the conversion of reduced glutathione (GSH) to its disulfide form, with concomitant consumption of O2 and release of H2O2. The ratio of GSH consumed to H2O2 produced was determined to be 2:1. At 25 degrees C, the optimum pH for the oxidation of GSH was 5.5. Under these conditions, the enzyme had a Michaelis constant of 0.3 mM for GSH. Other low molecular weight thiol compounds (cysteine, dithiothreitol, and 2-mercaptoethanol) were also oxidized, but the Michaelis constants for these substrates were substantially higher than that for GSH under identical conditions of temperature and pH. The rate of reactivation of reductively denatured ribonuclease A was enhanced by the presence of sulfhydryl oxidase, indicating that the latter is capable of oxidizing protein-associated thiol groups. The UV-visible spectrum of sulfhydryl oxidase solution had absorbance maxima at 274, 364.5, and 442.5 nm and was otherwise characteristic of the spectra of known flavoproteins.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 26:7363-7371(1987) [PubMed] [EuropePMC]

• A sulfhydryl oxidase from chicken egg white.

  Hoober K.L., Joneja B., White H.B., Thorpe C.

  A dimeric glycoprotein containing one FAD per approximately 80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/ ... >> More

  A dimeric glycoprotein containing one FAD per approximately 80,000 Mr subunit has been isolated from chicken egg white and found to have sulfhydryl oxidase activity with a range of small molecular weight thiols. Dithiothreitol was the best substrate of those tested, with a turnover number of 1030/min, a Km of 150 microM, and a pH optimum of about 7.5. Oxidation of thiol substrates generates hydrogen peroxide in aerobic solution. Anaerobically, the ferricenium ion is a facile alternative electron acceptor. Reduction of the oxidase with dithionite or dithiothreitol under anaerobic conditions yields a two-electron intermediate (EH2) showing a charge transfer band (lambdamax 560 nm; epsilonobs 2.5 mM-1 cm-1). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons. Borohydride and catalytic photoreduction give the same spectral changes. EH2, but not the oxidized enzyme, is inactivated by iodoacetamide with alkylation of 2.7 cysteine residues/subunit. These data indicate that the oxidase contains a redox-active disulfide bridge generating a thiolate to oxidized flavin charge transfer complex at the EH2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH2-like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed. << Less

  J Biol Chem 271:30510-30516(1996) [PubMed] [EuropePMC]