The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs." The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake ""respiratory burst"" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo."