Coupled copper pairs

Binding of NO to the spin-coupled copper pair of tyrosinase (Schoot Uter-kamp and Mason, 1973 Malmstrom, 1978) yields a dipolar coupled pair in which both copper ions bind NO the EPR spectra of the NO-complexed enzyme exhibits both broadened = 1 transitions and Sj = 2 transitions. 

Thereafter, crystals were brought back to the aerobic 25% MPD solution, buffered with 50 mAf sodium phosphate, pH 5.5. This procedure is based on Avigliano et al. s (157) method of preparing T2D ascorbate oxidase in solution and was modified by Merli et al. (159) for use with ascorbate oxidase crystals. The 2.5-A-resolution X-ray structure analysis by difference-Fourier techniques and crystallographic refinement shows that about 1.3 copper ions per ascorbate oxidase monomer are removed. The copper is lost from all three copper sites of the trinuclear copper species, whereby the EPR-active type-2 copper is the most depleted (see Fig. 10). Type-1 copper is not affected. The EPR spectra from polycrystalline samples of the respective native and T2D ascorbate oxidase were recorded. The native spectrum exhibits the type-1 and type-2 EPR signals in a ratio of about 1 1, as expected from the crystal structure. The T2D spectrum reveals the characteristic resonances of the type-1 copper center, also observed for T2D ascorbate oxidase in frozen solution, and the complete disappearance of the spectroscopic type-2 copper. This observation indicates preferential formation of a Cu-depleted form with the holes equally distributed over all three copper sites. Each of these Cu-depleted species may represent an anti-ferromagnetically coupled copper pair that is EPR-silent and that could explain the disappearance of the type-2 EPR signal. 

A number of cases have been reported in the literature where the exchange between pairs of ions in a lattice is piedominant, i.e., ions aie exchange coupled in pairs because of their proximity. An example of this is copper acetate 168), where the copper ions occur in pairs which are relatively close, so that the spins of pairs of copper ions are coupled together to form a singlet state (paired spins) and a triplet state (unpaired spins). Resonance measurements 158) permit determination of the magnitude and sign of J. 

The 46-kDa monomeric tyrosinase of Neurospom contains a pair of spin-coupled Cu(II) ions.568 569 The structure of this copper pair (type 3 copper) has many properties in common with the copper pair in hemo-cyanin.569a For example, in the absence of other substrates, tyrosinase binds 02 to form "oxytyrosinase," a compound with properties resembling those of oxyhemocyanin and containing a bound peroxide dianion.569... 

Fig. 5. Schematic representation of the interrelationships between the metal centers at the active site of cytochrome c oxidase showing an electronically coupled iron-copper pair (left) and an electronically and magnetically coupled iron-copper pair (right) which interact weakly with each other (—).

Copper-proteins are wide-spread in both animals and plants and have been related to many metabolic processes, as oxygen transport, electron transfer and hydroxylation Copper-containing sites are usually classified in three different types " the type 1, or blue center is characterized by a combination of properties that has not yet been reproduced in model complexes (an intense absorption band at 600 nm, a very small copper hyperfine coupling constant A and a high positive redox potential for the Cu(II)/ Cu(I) couple) the type 2, or non-blue center has properties comparable to those of low molecular weight cupric complexes the type 3 consists of an antiferromagnetically coupled copper(II) pair. 

C (66). If electron transfer from type 1 to type 3 copper couples the two halves of the enzyme cycle, as proposed for laccase, then this intramolecular redox reaction must be extremely rapid to account for the effects of trace dioxygen on the reduction of the type 1 copper. Consequently, despite the fact that an ambiguous assignment of a type 1 to type 3 transfer is not possible in this example, facile intramolecular electron transfer processes probably ensure a rapid distribution of electrons among the type 1 and type 3 copper centers, at least in some of the enzyme molecules. The equilibrium distribution, and quite conceivably the relative rates of approach to this state, should be influenced by the oxidation-reduction potentials, which, as described earlier in this chapter (Figure 5), favor electron occupancy of the type 3 copper pairs at 10.0°C. 

Cytochrome c oxidase (COX) is the terminal enzyme in the respiratory system of most aerobic organisms and catalyzes the four electron transfer from c-type cytochromes to dioxygen (115, 116). The A-type COX enzyme has three different redox-active metal centers A mixed-valence copper pair forming the so-called Cua center, a low-spin heme-a site, and a binuclear center formed by heme-fl3 and Cub. The Cua functions as the primary electron acceptor, from which electrons are transferred via heme-a to the heme-fl3/CuB center, where O2 is reduced to water. In the B-type COX heme-u is replaced by a heme-fo center. The intramolecular electron-transfer reactions are coupled to proton translocation across the membrane in which the enzyme resides (117-123) by a mechanism that is under active investigation (119, 124—126). The resulting electrochemical proton gradient is used by ATP synthase to generate ATP. 

Magnetic susceptibility measurements carried out on (15) in the temperature range 5 to 300 K are consistent with a system composed of an antiferromagnetically coupled copper(II) pair and a magnetically independent third copper(II) ion. The macrocyclic complex therefore reproduces qualitatively features of the oxidase active site and the observed 2J value of -192 cm indicates the presence of a moderate antiferromagnetic interaction within the dinuclear moiety, albeit weaker than that of the protein type-3 site which is diamagnetic even at room temperature. 

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