Coupled binuclear copper proteins,

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... 

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

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. 

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

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(106) ligated by histidine residues, with a sideways / 2-v2 V2 peroxo group bound to both Cu11 centers (104), which superseded the previous model (107). 

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. 

One of the major goals of studying active sites in copper proteins has therefore been to understand the spectroscopic features associated with the active site. This has led to a classification of three general types of copper protein active sites based on their unique spectral features Blue copper, normal copper and coupled binuclear copper. An additional class of copper proteins, the multi-copper oxidases, contains a combination of these three types of copper active sites. A reasonably firm understanding of the optical and EPR spectra of a number of copper proteins has now been achieved1,2K This article presents an overview of these electronic spectral features and their relationship to geometric and electronic structure. 

Chemical and spectral studies have indicated that in addition to bridging exogenous ligands, there is an endogenous protein bridge at the coupled binuclear copper site. Evidence for this is seen with the half-met derivative and the binuclear cupric derivatives met and dimer. 

When the coupled binuclear copper site in the protein becomes structurally characterized by x-ray crystallography, single-crystal spectroscopy should provide a more detailed picture of its electronic structure and bonding, as has been achieved for the Blue Copper site described in the previous section. 

Fig. 42. Spectroscopically effective active site representations of the coupled binuclear copper site (left) and the type 3 site in Rhus laccase (right) where OR and R represent endogenous protein bridges in the respective sites...

Lerch, K., and German, U. A. (1988). Evolutionary relationships among copper proteins containing coupled binuclear copper sites. In Oxidases and Related Redox Systems (K. T. S., Mason, H. S., and Morrison, M., Eds.), pp. 331-348. A. R. Liss, New York. 

Most of the synthetic models we found in our literature survey are concerned with hemocyanine, the oxygen transport protein in arthropods and molluscs, and tyrosinase, which catalyzes the two-electron oxidation of phenolic compounds. Both proteins contain a coupled binuclear copper active site, a Type III copper centre,"which reversibly binds dioxygen as peroxide bridging between the two copper ions" [153]. The Cu-Cu distance is of the order of 300-400 pm, and a tetragonal coordination is achieved with donor nitrogen atoms of imidazole ligands from histidine [142]. 

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. 

---