A sulfhydryl oxidase from chicken egg white

Area of Science:

• Biochemistry
• Enzymology

Background:

• A dimeric glycoprotein with sulfhydryl oxidase activity was isolated from chicken egg white.
• The enzyme contains flavin adenine dinucleotide (FAD) and acts on small molecular weight thiols.

Purpose of the Study:

• To characterize the kinetic and spectral properties of the isolated sulfhydryl oxidase.
• To elucidate the enzyme's catalytic mechanism and active site features.

Main Methods:

• Enzyme isolation and purification from chicken egg white.
• Enzymatic assays using various thiol substrates (e.g., dithiothreitol) to determine kinetic parameters (turnover number, Km, pH optimum).
• Spectroscopic analysis (UV-Vis) to characterize reaction intermediates and electron acceptors (ferricenium ion, dithionite, borohydride, photoreduction).
• Chemical modification studies using iodoacetamide to probe the active site.

Main Results:

• Dithiothreitol was identified as an optimal substrate with high turnover and a Km of 150 microM at pH 7.5.
• The enzyme generates hydrogen peroxide during aerobic thiol oxidation and utilizes ferricenium ion as an alternative electron acceptor anaerobically.
• A two-electron reduced intermediate (EH2) with a charge transfer band at 560 nm was observed, requiring four electrons for complete reduction.
• EH2 intermediate is inactivated by iodoacetamide, indicating alkylation of cysteine residues and involvement of a disulfide bridge.

Conclusions:

• The sulfhydryl oxidase contains a redox-active disulfide bridge crucial for its activity.
• A thiolate-to-flavin charge transfer complex is formed at the EH2 level.
• Sulfite treatment cleaves the disulfide bridge, yielding a stable EH2-like species.
• The enzyme exhibits functional similarities to the pyridine nucleotide-dependent disulfide oxidoreductase family.