Functional Changes in the Family of Type 3 Copper Proteins During Evolution

When life developed on earth, it happened in a reducing atmosphere where oxygen was only a trace element. Thus, the first cells and organisms produced metabolites necessary for life under anaerobic conditions. Some 3500 million years ago some bacteria began to meet their energy needs by photosynthesis. Dioxygen as well as its highly reactive derivatives, hydrogen peroxide, superoxide anions, and hydroxyl radicals, were released as by-products and became a severe poison for the hitherto anaerobic cells. To cope with these highly reactive oxygen species and minimize the damage caused by them, different kinds of enzymes emerged. These enzymes, such as superoxide dismutase, catalase, peroxidase, monooxygenases, and dioxygenases, react with oxygen to yield harmless end products. Many of these enzymes, such as tyrosinase (EC 1.14.18.1), incorporate oxygen into organic compounds by hydroxylation. This enzyme catalyzes two reactions, the hydroxylation of phenolic compounds in the ortho position (cresolase activity) and subsequently oxidation of diphenolic products (catecholase activity). Tyrosinase as well as catecholoxidase (EC 1.10.3.1), which catalyzes only the oxidation, belong to the group termed phenoloxidases. Their presence in all phyla of living organisms demonstrates their early origin in the history of life. Tyrosinases and catecholoxidases bind oxygen at copper-containing active sites, called type 3 copper sites. This distinction was originally based on their specific EPR spectra. 5] Their active site is a binuclear copper center consisting of two copper atoms, CuA and CuB, each coordinated by three histidines. The histidines are provided by two pairs of -helices forming a four -helix bundle motif (Figure 1). 7] Upon oxygen binding, a change of oxidation state is observed from Cu to Cu . 8] The type 3 copper-protein family includes not only tyrosinases and catecholoxidases but also hemocyanins, which are found extracellularly in the hemolymph of various mollusks and arthropods. Hemocyanins are responsible for the precise delivery of oxygen, in analogy to the hemoglobins. 9±11] Tyrosinases and hemocyanins have been investigated independently over the years, although comparative data have been reported. 10±15] The increasing volume of sequences and biophysical/biochemical data have suggested a study of both proteins, in a comparative way. Such comparative studies of type 3 copper protein sequences have revealed the existence of two different protein classes, which differ in their CuA and CuB environments. 11, 16, 17] CuA denotes the copper binding site closer to the N terminus, whereas CuB is the copper binding site closer to the C terminus. Figure 1. Structure of monoand binuclear copper centers. i) Binuclear copper centers (orange) bind dioxygen (red) in a TMside-on∫ coordination between two copper atoms as shown by a bioinorganic complex (ia) and the active site of the hemocyanin of the horseshoe crab Limulus polyphemus (ib). 30] ii) Crystallographic structure of a bioinorganic complex (iia), which is equivalent to a half active site of Limulus polyphemus hemocyanin (iib). It reveals that even a mono copper center can bind oxygen in a TMside-on∫ coordination. To date no protein has been described containing such a mononuclear active site.