A sulfhydryl oxidase from chicken egg white.
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.
Hoober
K L
KL
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA. cthorpe@strauss.udel.edu
Joneja
B
B
White
H B
HB
Thorpe
C
C
D000818:Animals; D002645:Chickens; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000378:metabolism; D004529:Egg White; D005420:Flavoproteins / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D010063:Ovum / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013447:Sulfites / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
D004220:Disulfides; D004229:Dithiothreitol; D005420:Flavoproteins; D010088:Oxidoreductases; D013447:Sulfites; D050862:Oxidoreductases Acting on Sulfur Group Donors