A Sulfhydryl Oxidase from Chicken Egg White

Abstract

A dimeric glycoprotein containing one FAD per ϳ80,000 M r 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 K m of 150 M, 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 (EH 2 ) showing a charge transfer band ( max 560 nm; ⑀ obs 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. EH 2 , 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 EH 2 level. Sulfite treatment does not form the expected flavin adduct with the native enzyme but cleaves the active site disulfide, yielding an air-stable EH 2 -like species. The close functional resemblance of the oxidase to the pyridine nucleotide-dependent disulfide oxidoreductase family is discussed.Sulfhydryl oxidases catalyze the oxidation of sulfhydryl groups to disulfides according to the following general reaction.They have been purified from a number of microbial and mammalian sources, but many questions concerning their mechanism and functional roles remain unresolved. Iron-dependent sulfhydryl oxidases have been found in bovine milk (1-3), in kidney (4), and in pancreatic zymogen granules (5). Coppercontaining sulfhydryl oxidases have been described from kidney and small intestine (6) and skin (7,8).A second distinct class of sulfhydryl oxidase is provided by FAD-dependent enzymes isolated from both rat seminal vesicles and fungal sources (9 -11). These careful studies show a range of diverse subunit composition and substrate specificity but clearly implicate the flavin cofactor in catalysis (9, 10). The present work reports the discovery and characterization of a flavoprotein sulfhydryl oxidase in chicken egg white.Our study began with the observation that egg white contains not only riboflavin (in the form of riboflavin-binding protein; Ref. 12) but also measurable levels of FAD (13). While the function of riboflavin-binding protein in the transport and storage of vitamin B 2 is well known (14), the reason for the presence of FAD in egg white was obscure. We first purified the FAD-containing protein by following its yellow color and subsequently identified it as a sulfhydryl oxidase active with a wide range of thiol substrates. This paper presents strong evidence for the involvement of a catalytically important disulfide as a second redox-active moiety in these flavin de...

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

“…Enzyme assays, using a Clarke-type oxygen electrode or discontinuous sampling using DTNB, were performed as described previously (26,30,31). Anaerobic manipulations and titrations were as described earlier (26,32). Photoreductions were performed in semi-micro anaerobic cuvettes immersed in ice-water using a 150 W flood lamp, in Tris buffer containing 1.0 µM 5-deazaflavin and 1 mM EDTA.…”

“…Evidently catalytic interactions between thiols and flavins have evolved independently multiple times. Prior experience with the pyridine nucleotide disulfide oxidoreductase family (23,45) and with avian QSOX (26,27), lead to the expectation that a thiolate-oxidized flavin charge-transfer complex would be observed on 2-electron reduction of Erv2p. Possible reasons why such absorbance bands have not been observed in native Erv2p are outlined below.…”

“…Kaiser and coworkers suggest that another ER protein may be the real physiological mediator for Erv2p, or a factor, which facilitates the redox interaction between PDI and Erv2p, may be missing from these in vitro experiments (24). Adding to the uncertainties concerning the catalytic specificity of Erv2p, we currently lack important basic information concerning the redox behavior of Erv2p to compare to the emerging data for human ALR (25), the plant mitochondrial AtErv1p (8,9), and the avian QSOX1 (26)(27)(28)(29). A comparison of the properties of these three rather diverse representatives of the ERV/ALR family of sulfhydryl oxidases will help clarify the basic features of flavinlinked catalysis in all of them.…”

“…Visible and ultraviolet spectra were recorded on HewlettPackard 8452A or 8453 diode array spectrophotometers. Enzyme assays, using a Clarke-type oxygen electrode or discontinuous sampling using DTNB, were performed as described previously (26,30,31). Anaerobic manipulations and titrations were as described earlier (26,32).…”

The Potentiation Role of Hepatopoietin on Activator Protein-1 Is Dependent on Its Sulfhydryl Oxidase Activity

“…Recently, it was reported that the ERV1/HPO family belongs to sulfhydryl oxidase (SOX) participating in disulfide bond formation (12-17). The SOX proteins contain a conserved CXXC motif and a non-covalent FAD adjacent to CXXC, which are vital to their catalytic activity (18,19). Sulfhydryl oxidases generally form dimers in vivo and catalyze the oxidation of sulfhydryl groups to disulfides according to the general reaction 2R-SH ϩ O 2 3 RSSR ϩ H 2 O 2 (18), which is different from the flavoprotein families (20).…”