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Members of the multicopper oxidase (MCO) family of enzymes can be classified by their substrate specificity; for example, ferroxidases oxidize ferrous iron, ascorbate oxidases oxidize ascorbate, and laccases oxidize aromatic substrates such as diphenols. Our previous work on an insect multicopper oxidase, MCO1, suggested that it may function as a ferroxidase. This hypothesis was based on three lines of evidence: RNAi-mediated knock down of Drosophila melanogaster MCO1 (DmMCO1) affects iron homeostasis, DmMCO1 has ferroxidase activity, and DmMCO1 has predicted iron binding residues. In our current study, we expanded our focus to include MCO1 from Anopheles gambiae, Tribolium castaneum, and Manduca sexta. We verified that MCO1 orthologs have similar expression profiles, and that the MCO1 protein is located on the basal surface of cells where it is positioned to oxidize substrates in the hemolymph. In addition, we determined that RNAi-mediated knock down of MCO1 in A. gambiae affects iron homeostasis. To further characterize the enzymatic activity of MCO1 orthologs, we purified recombinant MCO1 from all four insect species and performed kinetic analyses using ferrous iron, ascorbate and two diphenols as substrates. We found that all of the MCO1 orthologs are much better at oxidizing ascorbate than they are at oxidizing ferrous iron or diphenols. This result is surprising because ascorbate oxidases are thought to be specific to plants and fungi. An analysis of three predicted iron binding residues in DmMCO1 revealed that they are not required for ferroxidase or laccase activity, but two of the residues (His374 and Asp380) influence oxidation of ascorbate. These two residues are conserved in MCO1 orthologs from insects and crustaceans; therefore, they are likely to be important for MCO1 function. The results of this study suggest that MCO1 orthologs function as ascorbate oxidases and influence iron homeostasis through an unknown mechanism.

Multicopper ferroxidases catalyze the oxidation of ferrous iron to ferric iron. In yeast and algae, they participate in cellular uptake of iron; in mammals, they facilitate cellular efflux. The mechanisms of iron metabolism in insects are still poorly understood, and insect multicopper ferroxidases have not been identified. In this paper, we present evidence that Drosophila melanogaster multicopper oxidase-1 (MCO1) is a functional ferroxidase. We identified candidate iron-binding residues in the MCO1 sequence and found that purified recombinant MCO1 oxidizes ferrous iron. An association between MCO1 function and iron homeostasis was confirmed by two observations: RNAi-mediated knockdown of MCO1 resulted in decreased iron accumulation in midguts and whole insects, and weak knockdown increased the longevity of flies fed a toxic concentration of iron. Strong knockdown of MCO1 resulted in pupal lethality, indicating that MCO1 is an essential gene. Immunohistochemistry experiments demonstrated that MCO1 is located on the basal surfaces of the digestive system and Malpighian tubules. We propose that MCO1 oxidizes ferrous iron in the hemolymph and that the resulting ferric iron is bound by transferrin or melanotransferrin, leading to iron storage, iron withholding from pathogens, regulation of oxidative stress, and/or epithelial maturation. These proposed functions are distinct from those of other known ferroxidases. Given that MCO1 orthologues are present in all insect genomes analyzed to date, this discovery is an important step toward understanding iron metabolism in insects.