A sulfhydryl oxidase from chicken egg white

A dimeric glycoprotein containing one FAD per similar to 80,000 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 mu 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 (lambda(max) 560 nm; epsilon(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.