Function of dopachrome oxidoreductase and metal ions in dopachrome conversion in the eumelanin pathway

The conversion of dopachrome (DC) in the eumelanin pathway has been analyzed to determine the specific product and the role of enzyme control. 5,6-Dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) were quantitated by HPLC with fluorescent detection, after DC incubation with heated and unheated preparations of B-16 melanoma derived dopachrome oxidoreductase (DCOR). The enzyme-catalyzed reaction produced DHICA as the major product, while DHI formed with the spontaneous reaction. It had originally been suggested that the major product of DC conversion was DHI, with DHICA being formed as a minor product of this conversion [Raper, H.S. (1927) Biochem. J. 21, 89-96]. Copper, nickel, and cobalt ions promoted conversion of DC, with nickel simulating DCOR activity. Removal of free ions from unheated DCOR did not alter DC conversion. We conclude that the major product of DC conversion is DHICA and that DCOR is responsible for this conversion.