Hemocyanin active site

The structure of the ascorbate oxidase tricopper site is illustrated in Fig. 45. The three copper atoms form an almost equilateral triangle of sides ca. 3.7 A. The Cul and Cu2 atoms are bridged by OH" or O " and make up the EPR-silent type 3 pair each copper atom is coordinated to three histidine residues and the Cu—N(His) distances are all comparable and unexceptional. In contrast to the hemocyanin active site (Section IVD), the copper ions have approximately tetrahedral coordination geometry and are not in identical environments. The third copper ion is coordinated to two histidine residues and to either hydroxide or water. There is no evidence for a fJ-s-OH or fiyO donor at the center of the cluster (and the Cu—Cu distances are too long to support such a bridge). 

X-ray analysis of both copper(I) (e.g. 9) and copper(II) complexes, independent synthesis, and ammonolysis to the free ligands 35 and 36, confirmed the hydroxylation pathway.42 02-binding and hydroxylation in complex 10 were shown to be sensitive to electronic effects of the para-substituent (X = OMe, Me, C02Me, N02>. Tyrosinase, which contains a dinuclear copper active site strongly resembling the hemocyanine active site, binds O2 reversibly and activates O2 for arene hydroxylation (stoichiometry Cu O2 = 2 These are key features observed in the dinuclear copper complexes shown in... 

Ross, P. K., and E. I. Solomon. 1991. An Electronic Structural Comparison of Cooper-Peroxide Complexes of Relevance to Hemocyanin and Tyrosinase Active Sites. J. Am. Chem. Soc. 113, 3246. 

Senozan, N. et al. (1981) Hemocyanin of the giant keyhold limpet, Megathura crenulata. In Invertebrate Oxygen Binding Proteins Structure, Active Sites, andFunction (J. Lamy, and J. Lamy, eds.), pp. 703-717. Dekker, New York. 

The type 1-3 terminology to distinguish different Cu protein active sites remains extremely useful. Sub-groupings are appearing however in all three categories particularly in the case of the binuclear (EPR inactive) type 3 centers. Thus, in the recently determined X-ray crystal structure of ascorbate oxidase the type 3 and type 2 centers are present as a single trimer unit [. A discrete binuclear type 3 center is, however, retained in hemocyanin [6]. 

Evidence tom a variety of sources indicates that the active site of tyrosinase is very similar to that of hemocyanin, a dioxygen-binding protein found in molluscs and arthropods (15,16). This type of active site contains two copper ions, which are cuprous in the deoxy state, and which reversibly bind dioxygen, forming the oxy form of the enzyme or protein in which a peroxy ligand bridges between two cupric ions. 

Hemocyanin [30,31], tyrosinase [32] and catechol oxidase (2) [33] comprise this class of proteins. Their active sites are very similar and contain a dicopper core in which both Cu ions are ligated by three N-bound histidine residues. All three proteins are capable of binding dioxygen reversibly at ambient conditions. However, whereas hemocyanin is responsible for O2 transport in certain mollusks and arthropods, catechol oxidase and tyrosinase are enzymes that have vital catalytic functions in a variety of natural systems, namely the oxidation of phenolic substrates to catechols (Scheme 1) (tyrosinase) and the oxidation of catechols to o-quinones (tyrosinase and catechol oxidase). Antiferromagnetic coupling of the two Cu ions in the oxy state of these metalloproteins leads to ESR-silent behavior. Structural insight from X-ray crystallography is now available for all three enzymes, but details... 

As the focus of this review is on copper-dioxygen chemistry, we shall briefly summarize major aspects of the active site chemistry of those proteins involved in 02 processing. The active site structure and chemistry of hemocyanin (He, 02 carrier) and tyrosinase (Tyr, monooxygenase) will be emphasized, since the chemical studies described herein are most relevant to their function. The major classes of these proteins and their origins, primary functions, and leading references are provided in Table 1. Other classes of copper proteins not included here are blue electron carriers [13], copper-thiolate proteins (metallothioneines) [17], and NO reductases (e.g., nitrite [NIR] [18] or nitrous oxide [19]). 

The deoxy forms of hemocyanins are colorless, as a result of their 3d ° dicopper(I) centers. Although chemical and x-ray absorption spectroscopic studies had shed considerable light on the nature of the deoxy-He dicopper binding site, there now exist two x-ray crystal structures, the first on the the spiny lobster He, Panulirus interruptus [23], and a recent one of the horseshoe crab Limulus II protein [24], The structures exhibit rather different active-site characteristics, and since the former was crystallized at low pH and possesses rather odd copper coordination, the latter Limulus II structure is probably representative. It indicates that the two Cu(I) ions are 4.6 A apart, each found in a trigonal-planar coordination environment with Cu-NMs bond distances of about 2.0 A (Figure 1). Intersubunit 02 binding cooperative effects are probably initiated and trans-... 

Figure 5.1 Schematic representations of selected active sites of the copper proteins plastocyanin [56] (type 1, a) galactose oxidase [57] (type 2, b) oxy hemocyanin [58] (type 3, c) ascorbate oxidase [10] (type 4, or multicopper site, d) nitrous oxide reductase [59] (CuA site, e) cytochrome c oxidase [15]...

The interest in catechol oxidase, as well as in other copper proteins with the type 3 active site, is to a large extent due to their ability to process dioxygen from air at ambient conditions. While hemocyanin is an oxygen carrier in the hemolymph of some arthropods and mollusks, catechol oxidase and tyrosinase utilize it to perform the selective oxidation of organic substrates, for example, phenols and catechols. Therefore, establishment of structure-activity relationships for these enzymes and a complete elucidation of the mechanisms of enzymatic conversions through the development of synthetic models are expected to contribute greatly to the design of oxidation catalysts for potential industrial applications. 

The general feature of the active site of the purple hemocyanin can be elucidated as follows. The purple hemocyanin is an equilibrium mixture of approximately 60% of a slightly deformed species (abbreviated as A) and approximately 40% of a rather seriously deformed species (abbreviated as B). The species A exhibits the Raman peak for the O—O stretching vibration at around 750 cm-1, being ESR-inactive and responsible for the purple color. Though the species turns red upon the addition of NCS-, the oxidation state of the two coppers at the active site probably is kept as Cu(II) in an ESR-inactive structure. To satisfy this requirement, imidazole cannot be the bridg-... 

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