TERWILLIGER, N*; RYAN, M; JAENICKE, E; DECKER, H: Evolution of Copper-Oxygen Proteins: Hemocyanins Function as Phenoloxidases

Hemocyanins from arthropods are well known as oxygen transport molecules, but under some conditions they can function as phenoloxidases. Phenoloxidases and tyrosinases (EC 1.14.18.1) are found in fungi and plants as well as animals, and they are involved in wound healing, skin pigmentation, the browning of plants and the initiation of exoskeleton hardening or sclerotization. Published sequences of phenoloxidases from arthropods show they are related to arthropod hemocyanins. Here, we show that hemocyanins from two ancient chelicerates, the horseshoe crab, Limulus polyphemus, and the tarantula, Eurypelma californicum, have phenoloxidase activity induced by submicellar concentrations of SDS. The enzyme activity is restricted to only a few of the heterogeneous subunits, those that share similar topological positions as linkers in the four- to eight-hexamer quaternary structures. No other phenoloxidase activity has been found in the hemolymph of these chelicerates, suggesting that their hemocyanins are involved in both oxygen transport and in the primary immune response and sclerotization of the exoskeleton. In contrast, hemolymph from a more recent crustacean, Cancer magister, has two different molecules with phenoloxidase activity. One is hemocyanin, while the predominant phenoloxidase is a separate protein. The linker subunits in chelicerate hemocyanin that have phenoloxidase activity may have evolved into separate diphenoloxidases in crustaceans, whose two-hexamer hemocyanins lack these linker subunits. This work was supported by NSF 9984202 (NBT) and the NMFZ Mainz (HD).