Copper center, type

Electron transfer (continued) intramolecular, from type-1 copper center to trinuclear copper center blue copper oxidases, 40 175-178 iron-sulfur proteins, 47 405, 474-479 kinetic model, flavocytochrome bj, 36 282-283... 

A new putative member of the blue multi-copper oxidases has been isolated using the Escherichia coli yacK gene. Six copper ions per polypeptide chain were determined and assigned to two type 1 copper centers and further one type 2 and one type 3 copper. Phenoloxidase and ferroxidase properties were ascertained98 A new copper containing nitrite reductase was purified from a halophilic archaeon and the ligands to type 1 and type 2 coppers in the sequence were... 

Several copper-containing NiRs have been identified, but the most extensive structural and mechanistic studies have focused on the enzyme from Achromobacter cycloclastes (17-25). A 2.3-A resolution X-ray crystal structure for this NiR in its oxidized form at pH 5.2 has been reported (17), and a representation of the active site is shown in Figure 1. Each monomer in the trimeric protein contains two copper ions, one of which (Cu-1) is ligated to a cysteine, a methionine, and two histidine residues in a geometry similar to that of type 1 copper centers in proteins such as plastocyanin (26). The second type 2 copper ion in NiR (Cu-2) is only 12.5-A distant from the first and is bound to three histidine imidazoles (two from one monomer, the third from an associated subunit) and a fourth small ligand in an unusual tetrahedral arrangement. The... 

B. Intramolecular Electron Transfer from the Type-1 Copper Center to the Trinuclear Copper Center... 

The number and type of copper centers, EPR parameters, and the two relevant absorption bands in the visible region for several representative members of the blue oxidases are listed in Table II. All laccases except that of the P. radiata enzyme contain four coppers per molecule with one type-1, one type-2, and one type-3 copper center. The EPR and absorption parameters resemble each other very much. Phlebia radiata laccase is supposed to contain only two coppers with one type 1, one type 2, and one PQQ per mole (70). This is quite unusual and will be discussed critically below, since it is possible that the copper content determined for this enzyme is inaccurate. Ascorbate oxidases have eight coppers per homodimer with two type-1, two type-2, and two type-3 copper centers. Ceruloplasmin typically contains six to seven copper ions per molecule with three type-1, one type-2, and one type-3 copper centers. It has also been proposed that there are only two type-1 copper ions and a new type-4 copper that is presumed to exhibit no EPR signal. In addition there is a variable content of chelatable copper. It is responsible for copper contents exceeding 6 coppers/mol but does not seem to be required for catalysis. It is now generally accepted that ceruloplasmin has three type-1 copper centers and the reason for this will be discussed below. 

Fig. 7. Stereo drawing of the region of the atomic model containing the type-1 copper center in domain 3 and the trinuclear copper center between domain 1 and domain 3.

The shortest distance between the type-1 copper center and the trinuclear copper center is 12.2 A. The His506-Cys507-His508 amino-acid sequence segment links the type-1 copper center and the type-3 copper center as a bridging ligand (see Fig. 7). Aspects of the intramolecular electron transfer between the two redox centers will be discussed in Section XII. 

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. 

As previously mentioned, the electron transfer from one-electron-reduced nitroaromates (ArN02 ), C02 , methyl viologen, lumi-flavin, or deazaflavin to the type-1 copper center (see Table VIII) takes... 

The finding from rapid-freeze-quench EPR experiments, that the reduction of the type 2 copper is slow compared with that of the type 1 copper, is analogous to the behavior noted for tree laccase at higher pH values (50). In this enzyme the slow reduction of the type 2 center is linked to the inhibition of the type 3 reduction. In ascorbate oxidase, however, reduction of the type 3 copper pairs proceeds despite the slow reduction of the type 2 copper, suggesting that the two electrons necessary for the proposed intramolecular reduction of the two type 3 copper pairs can be transferred from two of the three type 1 copper centers, without involving the type 2 center in any redox process. 

A remarkable feature of some copper-proteins is an intensive blue color based on the spectroscopic properties of the type 1 copper center. Type 1 copper centers show a characteristic absorption band at approximately 600 nm and an extinction coefficient exceeding 3000 1 mol-1 cm"1. In comparison, the extinction coefficient of the hexaquacopper(II) complex, [Cu(OH2)6]2+, whose blue color is much less intensive, is only 5-10 1 mol"1 cm"1 [6]. This difference in spectro-... 

Fig. 5. a The structure of plastocyanin from poplar, b Stick-and-ball model of the plasto cyanin type 1 copper center. From Lippard and Berg 1995 [6] with permission... 

Type 1 copper centers are found in small blue proteins, in blue oxidases and in nitrite reductase. In the latter, the distorted ligand geometry causes a shift in the absorption band resulting in a green instead of a blue enzyme [26,27]. 

The properties of the type 2 copper centers resemble those of copper(II) complexes [17]. This indicates that the geometric conditions are less critical than in type 1 copper centers. 

Small blue proteins are involved in various biochemical processes. Where their physiological function is known, it is that of single-electron transport proteins. The range of their redox potentials reaches from +183 mV (Halocyanin [18], + 184 mV Stellacyanin [68] to 680 mV (Rusticyanin [68, 69]) as compared to Cu2+/Cu+, E° = +153 mV. Very few redox proteins function in this range. This feature, and their characteristic blue color are the product of the type 1 copper center, the only redox-active group in these proteins. During electron... 

The first histidine residue is in strand 4. The other ligands are situated in the loop connecting strands 7 and 8. The copper atom is bound in a depression near the surface of the protein formed by strands 2 a, 4,7, and 8. The distorted tetrahedral stereochemistries of the type 1 copper centers differ slightly from protein to protein [20]. Although small, these differences are ultimately responsible... 

The type 1 copper center of the phytocyanins differs from that of the other small blue proteins in that the methionine ligand is replaced by a glutamine [24]. The redox potential of stellacyanin from Rhus vernicifera is 184 mV [68]. 

Rusticyanin s type 1 copper center with its ligands His 85, Cys 138, His 143, and Met 148 resembles those of the other small blue proteins. Although its tertiary structure is a /1-meander, there are distinct differences between the amino acid folding patterns of rusticyanin and the other small blue proteins. Plastocyanin contains 8 [22, 71], amicyanin 9 [20, 78], and rusticyanin 13 / -strands, respectively [112] (Fig. 16). 

The central copper ion is surrounded by 36 amino acids. These 36 residues include the 4 copper ligands, 7 glycins, as well as 20 hydrophobic residues. The remaining five residues are ambivalent two serines, one threonine, one glutamic acid, and one aspartic acid, both of which are protonated at these low pH values. Thus, the type 1 copper center is protected from the solvent by a hydro-phobic micro-environment, thereby achieving acid stability and a high redox potential [112],... 

The proteins treated in this section will only contain type 2 copper centers. Additional type 2 copper centers may be found in blue oxidases, in which they form trinuclear centers with type 3 copper ions, and in nitrite reductase. In nitrite reductase, the type 2 copper center is coupled to a type 1 copper center via a cysteine residue. 

Non-blue oxidases are members of the enzyme class of oxidases, which oxidize their substrates by subtracting electrons without the transferal of oxygen. Two electrons are transferred from the substrate to oxygen, which is reduced to hydrogen peroxide. In contrast to the blue oxidases, non-blue oxidases contain neither type 1 copper centers, which are responsible for the former s color, nor type 3. 

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