The earth’s atmosphere changed from anaerobic to aerobic as oxygenic photosynthetic organisms

as oxygenic photosynthetic organisms evolved. Organisms had to develop different strategies to handle a final product of photosynthesis, dioxygen (O2), as well as its highly reactive derivatives, superoxide anions, hydrogen peroxide and hydroxyl radicals. Several enzymes, such as superoxide dismutase, catalase and peroxidase, evolved that protected the organisms. A particular class of protein, the copper oxygenbinding proteins (COPs), is of special interest, since organisms used these proteins to help harness oxygen. Primitive COPs eventually developed multiple functions. COPs may first have become robbers in the form of phenoloxidases to keep the amount of free dioxygen low. The enzymatic action of phenoloxidase robs free dioxygen of its molecular status, inserting one of the oxygen atoms into a phenol and releasing the other as water. As organisms became larger and circulatory systems evolved in multicellular organisms, COPs along with iron oxygen-binding proteins became oxygen-transport proteins, storing dioxygen and moving it from places of high concentration to places of low concentration within the body. Thus, organisms used COPs both to protect themselves against the highly reactive atmospheric dioxygen and to utilize this energy source. These considerations are supported by two recent observations that may also provide new insight into the evolutionary strategies by which organisms deal with dioxygen. During the past few years, several sequences of phenoloxidases (EC 1.14.18.1, tyrosinase) from arthropods have been published which reveal that the phenoloxidases are related to the arthropod haemocyanins (Aspan et al., 1995; Fujimoto et al., 1995). During the same period, haemocyanins from arthropods and molluscs, well-known as oxygentransport proteins, were shown to function as phenoloxidases under some conditions (Zlateva et al., 1996; Salvato et al., 1998; Decker and Rimke, 1998). Phenoloxidase catalyzes the incorporation of oxygen into phenolic molecules in a two-step reaction (Salvato and Beltramini, 1990; Sanchez-Ferrer et al., 1995; Solomon et al., 1996): First, a monophenol is orthohydroxylated (monophenol oxidase activity), and the resulting o-diphenol is then oxidized to an o-quinone (catecholase activity) (Fig. 1). As a result of this reaction, one oxygen of the bound dioxygen is incorporated (Mason, 1955). Thus, the enzyme is an oxygenase as well as an oxidase. The phenoloxidase reaction is found in fungi and plants as well as in animals: phenoloxidase is involved in wound healing, in skin pigmentation and in the browning of fruits and vegetables (Prota, 1992; van Gelder et al., 1997). The reaction is thought to protect plants and animals against intruders by forming melanin to encapsulate the intruders or to create an impervious scab (Anderson, 1991; Ashida and Yamazaki, 1990; Sugumaran, 1990; Barret, 1991). In insects, phenoloxidase initiates sclerotization of the new exoskeleton after moulting. In all cases, monophenolic derivatives such as L-tyrosine are the primary substrates that are converted to dopaquinone derivatives. What do the two groups of copper oxygen-binding proteins, haemocyanins as oxygen-transport proteins and phenoloxidases as enzymes, have in common? They share several physico-chemical properties (Jolly et al., 1972; Lerch, 1981, 1987; Kuiper et al., 1980; Himmelwright et al., 1980; Salvato and Beltramini, 1990; Beltramini et al., 1990; Solomon et al., 1994, 1996; Ling et al., 1994), which led to the 1777 The Journal of Experimental Biology 203, 1777–1782 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2708