Copper is an essential cofactor in a diversity of biological oxidation-reduction reactions due to its existence in either a reduced (Cu+) or an oxidized (Cu2+) state [1–3]. Oxidized copper preferentially coordinates with oxygen in aspartic and glutamic acids or with the imidazole nitrogen group in histidines , allowing interactions with a wide spectrum of proteins. Copper-binding proteins are present in the three domains of life [4–7] and are divided into three classes based on their spectroscopic properties and geometric structure of the active site: type-1 or blue copper proteins, which are involved in electron-transfer (e.g., plastocyanin, azurin and halocyanin); type-2 or non-blue copper proteins, which form part of the oxidoreductase family (e.g., galactose oxidase) [1, 2, 8]; and the type-3 or binuclear copper protein family, which comprises genes encoding tyrosinase, tyrosinase-related proteins, catechol oxidase and hemocyanin.
Tyrosinases and catechol oxidases, both commonly known as phenoloxidases, are enzymes involved in the oxidation of phenolic compounds. Tyrosinases catalyse the o-hydroxylation of monophenols and oxidation of o-diphenols to o-quinones [9, 10], whereas catechol oxidases are only able to catalyse the oxidation of o-diphenols . Hemocyanins are mainly oxygen carrier proteins that under specific circumstances have enzymatic activity [6, 10, 12–14]. All type-3 copper proteins share a similar binuclear active site that is composed of two copper atoms [Cu(A) and Cu(B)], each of which is coordinated by three conserved histidine residues [11, 15]. Although type-3 copper proteins possess similar active sites in terms of overall structure and ability to bind molecular oxygen, they each differ in amino acid sequence [6, 10, 15, 16]. These amino acid differences affect the substrate-binding pocket and the accessibility of the substrate to the active site .
The differences in the active site of type-3 copper proteins are likely to have enabled the diversification of these proteins into a wide array of biological processes, including oxygen transport in molluscs and arthropods, innate immunity, wound healing and melanin pigment synthesis in a variety of metazoans, and the browning of fruits and vegetables [5, 10, 17–19]. Some of the chemical reactions catalysed by catechol oxidases and tyrosinases are thought to protect plants and animals by the formation of melanised structures that can encase foreign invaders [5, 19]. Tyrosinases also contribute to calcified structures, such as molluscan shells and arthropod exoskeletons [20–23].
Despite the broad distribution and importance of type-3 copper proteins, the evolution of this family has only been studied in detail in plants (catechol oxidases) and specific animal lineages, including chordates (tyrosinases and tyrosinase related-proteins), arthropods and molluscs (tyrosinases and hemocyanins) [10, 16, 24–31]. Molluscs and arthropods both possess tyrosinases and hemocyanins, however previous studies have shown that tyrosinases and hemocyanins differ considerably at the amino acid level between these organisms, indicating that both proteins evolved independently [16, 25, 26, 32, 33]. In chordates, gene structure and phylogenetic analyses suggest that tyrosinases arose from a single ancestral gene [24, 28, 34] that duplicated before the divergence of urochordate and vertebrate lineages , leading to genes encoding tyrosinase and tyrosinase related-proteins. The latter then duplicated early in vertebrate evolution, giving rise to tyrosinase related-proteins 1 and 2 [24, 27, 30]. In chordates, tyrosinase related-proteins are grouped under the tyrosinase gene family, although not all these genes have tyrosinase activity (e.g., tyrosinase related-protein 2 has dopachrome tautomerase activity and there is some evidence that it binds zinc rather than copper at the active site) . In plants, catechol oxidase genes display a dynamic and complex evolutionary pattern (e.g. soy bean has undergone extensive species-specific gene duplication and divergence) . Here, we analyse available genomic resources of diverse metazoan and other eukaryotic and prokaryotic species to reconstruct the origin and evolution of type-3 copper proteins and propose a new classification scheme for this protein family.