Binuclear Copper Proteins

Active Sites in Copper Proteins. An Electronic Structure Overview 

A series of hemocyanin and tyrosinase active site derivatives (Fig. 23) can be prepared61 66), allowing systematic variation of the binuclear copper active site and chemical perturbation for spectral studies. In the simplest derivative, met-apo, one copper has been removed and the remaining copper oxidized to the spectroscopically accessible Cu(II). Next in complexity is a mixed-valent binuclear copper site. The Cu(II), in this half-met derivative, exhibits open-shell d9 spectroscopic features and the Cu(I), though spectroscopically inaccessible, can still be studied by comparison to the met-apo derivative. Two derivatives have formally binuclear cupric sites met, which is EPR-non-detect-able, and dimer, which exhibits an intense broad EPR signal. Spectroscopic study of these derivatives has led to the present picture of the coupled binuclear copper protein active site shown at the bottom of Fig. 23. 

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Our earlier research on the coupled binuclear copper proteins generated a series of protein derivatives in which the active site was systematically varied and subjected to a variety of spectroscopic probes. These studies developed a Spectroscopically Effective Model for the oxyhemocyanin active slte.(l) The coupled binuclear copper active site in tyrosinase was farther shown to be extremely similar to that of the hemocyanlns with differences in reactivity correlating to active site accessibility, and to the monophenol coordinating directly to the copper(II) of the oxytyroslnase site.(2) These studies have been presented in a number of reviews.(3) In the first part of this chapter, we summarize some of our more recent results related to the unique spectral features of oxyhemocyanin, and use... 

Our own work in the area of aerobic oxidations was inspired by the exquisite research performed on the structure and reactivity of the binuclear copper proteins (7), hemocyanin and tyrosinase, and by the seminal contribution of Riviere and Jallabert (8). These two authors have shown that the simple copper complex CuCl - Phen (Phen = 1,10-phenanthroline) promoted the aerobic oxidation of benzylic alcohols to the corresponding aromatic aldehydes and ketones (Fig. 2). 

Figure 9. Coupled binuclear copper proteins ground- and excited-state spectral features.

Since the choice is difficult due to the huge amount of examples, the selection is based either on aesthetic criteria (77) and (78), or on important applications. In a review, Synthetic Models for Binuclear Copper Proteins, Sorrell reports his previous results with pyrazole ligands which mimics histidyl-imidazole <89T3>. 

The reaction of binuclear copper complexes with oxygen as models for tyrosinase activity was also markedly accelerated by applying pressure (106408 ). Tyrosinase is a dinuclear copper protein which catalyses the hydroxylation of phenols. This reaction was first successfully modeled by Karlin and co-workers (109), who found that an intramolecular hydroxylation occurred when the binuclear Cu(I) complex of XYL-H was treated with oxygen (Scheme 5). 

Since the first EPR work on Cu(II) ions in proteins in the late fifties193, a great many EPR investigations on copper-containing proteins have been reported194-198. For a classification of the copper proteins into type I (blue copper), type II (non-blue copper) and type III (binuclear cupric pair), the reader is referred to Fee197. ... 

The second class of dioxygen carriers is that of haemocyanins. These proteins, which contain a binuclear Cu(I) site (thus in the oxidized Cu(II) met form they belong to the so-called Type 3 copper proteins , which contain an EPR-silent dicopper active site), regulate dioxygen transport in the respiration of arthropods and molluscs. Figures 7 and 8 show... 

Table 1. Proteins Containing Coupled Binuclear Copper Active Sites and Their Functions...

The current chapter focuses on the electrochemistry of the ionic forms of copper in solution, starting with the potentials of various copper species. This includes the effect of coordination geometry, donor atoms, and solvent upon the electrochemical potentials of copper redox couples, specifically Cu(II/I). This is followed by a discussion of the various types of coupled chemical reactions that may contribute to the observed Cu(II/I) electrochemical behavior and the characteristics that may be used to distinguish the presence of each of these mechanisms. The chapter concludes with brief discussions of the electrochemical properties of copper proteins, unidentate and binuclear complexes. 

We first consider proteins containing a single type of a copper site starting with mononuclear ones. Subsequently, binuclear sites and multi-centered copper proteins will be discussed. 

Like hemerythrin, hemocyanin is an oxygen transport non-heme-containing protein found in some arthropods and molluscs (104,105). In the 02-bound form, hemocyanin contains an antiferromagnetically coupled binuclear copper(II) system (106) ligated by histidine residues, with a sideways / 2-v2 V2 peroxo group bound to both Cu11 centers (104), which superseded the previous model (107). 

Cytochrome c (see Fig. 4-18) is a soluble protein of the intermembrane space. After its single heme accepts an electron from Complex III, cytochrome c moves to Complex IV to donate the electron to a binuclear copper center. 

Tyrosinase is both an oxidase and a hydroxylase. Some other copper enzymes have only a hydroxylase function. One of the best understood of these is the peptidylglycine a-hydroxylating monoxygenase, which catalyzes the first step of the reaction of Eq. 10-11. The enzyme is a colorless two-copper protein but the copper atoms are 1.1 nm apart and do not form a binuclear center.570 Ascorbate is an essential cosubstrate, with two molecules being oxidized to the semidehydro-ascorbate radical as both coppers are reduced to Cu(I). A ternary complex of reduced enzyme, peptide, and 02 is formed and reacts to give the hydroxylated product.570 A related two-copper enzyme is dopamine (J-monooxygenase, which utilizes 02 and ascorbate to hydroxylate dopamine to noradrenaline (Chapter 25).571/572 These and other types of hydroxylases are compared in Chapter 18. 

Several diverse metal centres are involved in the catalysis of monooxygenation or hydroxylation reactions. The most important of these is cytochrome P-450, a hemoprotein with a cysteine residue as an axial ligand. Tyrosinase involves a coupled binuclear copper site, while dopamine jS-hydroxylase is also a copper protein but probably involves four binuclear copper sites, which are different from the tyrosinase sites. Putidamonooxin involves an iron-sulfur protein and a non-heme iron. In all cases a peroxo complex appears to be the active species. 

This protein contains a coupled binuclear copper site that appears to be very similar to that found in hemocyanin (Section 62.1.12.3.8).1399 Tyrosinase catalyzes the hydroxylation of monophenols, and also behaves as an oxidase in the oxidation of orfho-diphenols. The deoxy protein [copper(I)] binds dioxygen to give oxytyrosinase, which is a Cu11 peroxide species with antiferromagnetic coupling between the two Cu11 centres. The oxybinuclear site is diamagnetic to the most sensitive detectors. 

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