Copper Sites

The copper binding sites of tyrosinases share a high sequence homology with those of the hemocyanins, the oxygen carrier proteins of the molluscs and arthropods (Schoot-Uiterkamp and Mason 1973; van Gelder et al. 1997; Decker et al. 2007; Decker and Tuczek 2000; van Holde et al. 2001). A functional change in this protein family is proposed to have occurred during the course of evolution, from enzymatic oxygen detoxification towards oxygen transport (Jaenicke and Decker 2004).

The common feature of tyrosinases is a "type 3 copper center," a diamagnetic spin-coupled copper pair (Lerch 1995; Sánchez-Ferrer et al. 1995; García-Borrón and Solano 2002) (Table 13.1). Sequence alignments of many pro- and eukaryotic tyrosinases have shown that the copper binding regions are highly conserved. The signatures of CuA and CuB are H-x(n)-H-x(8)-H and H-x(3)-H-x(n)-H, respectively.

Each of the two metal atoms, CuA and CuB, at the active site are coordinated by three conserved histidines located in a "four a-helix bundle." During the catalytic cycle, the type 3 copper center can adopt different functional forms: the oxy-state [Cu(II)-O22--Cu(II)], the deoxy-state [Cu(I) Cu(I)], the half-met state [Cu(I) Cu(II)], and the met state [Cu(II)-OH--Cu(II)]. In the latter case, the two copper atoms are bridged by hydroxo ions. The valences of the two copper atoms change from Cu(I)

HO L-Tyrosine ho^^z-y-cooh

COOH

TYROSINASE --

L-DOPA

+ Cysteine (or Glutathione)

ONH,

COOH

Dopaquinone

COOH

I 5-S-Cysteinyldopa

HO COOH

S 1,4-Benzothiazinylalanine

HO HO

O HO

N COOH H

Leucodopachrome

PHAEOMELANIN

(yellow-red-brown)

HO HO

Dopachrome (red)

N COOH H

5,6-Dihydroxyindole Dihydroxyindole-2-carboxylic acid

Indole-5,6-quinone Indole-5,6-quinone carboxylic acid

N COOH H

EUMELANIN

(black-brown)

Fig. 13.2 Biosynthesis of melanin from tyrosine (modified from Kobayashi et al. 1995; Sanchez-Ferrer et al. 1995; Seo et al. 2003)

to Cu(II), which can be followed spectroscopically. In the oxy state, the molecular oxygen is reversibly bound as a peroxide between the two copper atoms in a "side-on" conformation. In the absence of any substrate, more than 85% of the enzyme is in the met state, which can be regarded as the resting form of tyrosinase. The current view is that both the met and the oxy states of tyrosinases enable diphenoloxidase activity, whereas the monohydroxylase reaction requires the oxy state.

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