Purification and characterization of hydroxyindole oxidase from the gills of Mytilus edulis.

An hydroxyindole oxidase has been purified 100-fold from the gill plates of Mytilus edulis. This preparation yields a single band on acrylamide gel electrophoresis and isoelectric focusing. Metal analysis and spectral data show that both heme iron and copper are present in the purified enzyme in a 1:1 molar ratio. Potassium cyanide, sodium azide, various copper chelators, and carbon monoxide inhibit the enzyme-catalyzed reaction. These inhibition studies, as well as the photoreversibility of the CO inhibition, lend support to the conclusion that metal ions function as necessary cofactors for substrate oxidation. The Mytilus gill enzyme oxidizes a variety of substrates, among them biogenic amines. For all substrates tested, 1 dioxygen molecule is consumed/mol of substrate oxidized. When p-coumaric acid is hydroxylated, caffeic acid is formed in quantitative yield. H2O2 has been identified as a second product of the oxidation reaction. The addition of catalase prior to the onset of reaction is inhibitory while H2O2 or ascorbate activate the enzyme. These findings suggest that the hydroxyindole oxidase may be a monooxygenase which does not require reduced pyridine nucleotide for reductive activation.