A SULFHYDRYL OXIDASE FROM CHICKEN EGG-WHITE

A dimeric glycoprotein containing one FAD per similar to 80,000 M(r) s ubunit has been isolated from chicken egg white and found to have sulf hydryl oxidase activity with a range of small molecular weight thiols, Dithiothreitol was the best substrate of those tested, with a turnove r 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 aerobi c solution, Anaerobically, the ferricenium ion is a facile alternative electron acceptor, Reduction of the oxidase with dithionite or dithio threitol under anaerobic conditions yields a two electron intermediate (EH(2)) showing a charge transfer band (lambda(max) 560 nm; epsilon(o bs) 2.5 mM(-1) cm(-1)). Complete bleaching of the flavin and discharge of the charge transfer complex require a total of four electrons, Bor ohydride and catalytic photoreduction give the same spectral changes, EH(2), but not the oxidized enzyme, is inactivated by iodoacetamide wi th alkylation of 2.7 cysteine residues/subunit. These data indicate th at the oxidase contains a redox-active disulfide bridge generating a t hiolate to oxidized flavin charge transfer complex at the EH(2) level, Sulfite treatment does not form the expected flavin adduct with the n ative enzyme but cleaves the active site disulfide, yielding an air-st able EH(2)-like species. The close functional resemblance of the oxida se to the pyridine nucleotide-dependent disulfide oxidoreductase famil y is discussed.