Ovothiol: a novel thiohistidine compound from sea urchin eggs that confers NAD(P)H-O2 oxidoreductase activity on ovoperoxidase

Abstract

Sea urchin eggs contain a small molecular weight heat-stable factor that confers cyanide-resistant NAD(P)H-O2 oxidoreductase activity on ovoperoxidase (Turner, E., Somers, C. E., and Shapiro, B. M. (1985) J. Biol. Chem. 260, 13163-13171), the enzyme responsible for cross-linking the extracellular protein coat (fertilization membrane) of the egg. Here we report the isolation of the active cofactor and its identification by ultraviolet, NMR, and mass spectroscopy as a new sulfur-containing amino acid derivative, 1-methyl-alpha N,alpha N-dimethyl-4-thiohistidine, or ovothiol. Ovothiol reacts slowly with atmospheric oxygen or rapidly with micromolar concentrations of H2O2 to form ovothiol disulfide, which is inactive as a cofactor for the ovoperoxidase NAD(P)H oxidase. Reduced active ovothiol is regenerated by treatment with disulfide reductants and shows significant differences in its ultraviolet and NMR spectra from oxidized ovothiol. The oxidoreductase activity of the ovoperoxidase/ovothiol system is similar to that previously characterized with crude cofactor preparations; it is greatly enhanced by Mn2+ and is relatively insensitive to CN-, compared to the peroxidase activity of ovoperoxidase. The ovothiol content of eggs is estimated as 1.8 pmol/egg or an intracellular concentration of 6.8 mM. This concentration exceeds the amount of reductant needed for the CN-(-)insensitive oxygen consumption following fertilization and used in the production of H2O2 for fertilization membrane cross-linking. Whether ovothiol is involved in the cross-linking reaction, protects the egg from damage from H2O2, or has another role in development remains unclear.