Blue copper proteins isolation

The binding of cyclic thioethers to metal centers has also led to the isolation of complexes in which the coordinative properties of the ligand do not lit the stereochemical preferences of the metal ion(s) (188), Thus, a series of macrocyclic thioether complexes incorporating unusual stereochemistries and/or oxidation states has been generated (188). This is linked to the biological activity of the blue copper proteins and model systems in which the coordination geometry about Cu(II) is strained [in an entatic state (.212,221)] such that the Cu(II)/(I) couple occurs at a particularly positive potential that is, the Cud) state is stabilized. The ability of cyclic thioethers to modify their coordination properties is inherent in this approach (76,108,111). 

This topic has been reviewed by Ingledew (55). The major components of the respiratory chain for T. ferrooxidans are a cytochrome oxidase of the Ci type, cytochromes c, and the blue copper protein rusticyanin. Initial electron transfer from Fe(II) to a cellular component takes place at the outer surface of the plasma membrane in the periplasmic space. The rate of electron transfer from Fe(II) to rusticyanin is too slow for rusticyanin to serve as the initial electron acceptor. Several proposals have been made for the primary site of iron oxidation. Ingledew (56) has suggested that the Fe(II) is oxidized by Fe(III) boimd to the cell wall the electron then moves rapidly through the polynuclear Fe(III) complex to rusticyanin or an alternative electron acceptor. Other proposals for the initial electron acceptor include a three-iron-sulfur cluster present in a membrane-bound Fe(II) oxidoreductase (39, 88), a 63,000 molecular weight Fe(II)-oxidizing enzyme isolated from T. ferrooxidans (40), and an acid-stable cytochrome c present in crude extracts of T. ferrooxidans (14). 

Single-crystal structural data have provided valuable information about blue copper proteins containing Type 1 Cu centres. Figure 28.10a shows a representation of the folded protein chain of spinach plastocyanin. The Cu(II) centre lies within a pocket in the chain, bound by a Cys, a Met and two His residues (Figure 28.10b) the S(Met) atom is significantly further away from the Cu(II) centre than is S(Cys). Figure 28.10c shows the backbone of the protein chain in azurin isolated from the bacterium Pseudomonas putida. The coordination environment of the Cu(II) centre resembles that in plastocyanin with Cu—S(Met) > Cu—S(Cys), but in addition, an O atom from an adjacent Gly residue is involved in a weak coordinate interaction (Figure 28.10d). Structural... 

SOD, isolated from bovine erythrocytes, is a blue-green protein due to the presence of copper, removal of which by treatment with EDTA results in loss of activity, which is restored by adding Cu2+ it also contains Zn2+, which does not appear to be at the active site. The enzyme, which is very stable in 9 M urea at neutral pH, consists of two identical subunits of molecular weight 16kDa held together by one or more disulphide bonds. The amino acid sequence has been established. 

In Section II we discussed the properties of certain proteins containing single or isolated and independent blue copper centers. With the exception of plastocyanin the biological function of these proteins is not known, but it is very likely that they participate in single electron transfer reactions. 

One of the most intensively studied copper proteins is caeruloplasmin but despite extensive knowledge of its structure its function remains obscure. Caeruloplasmin, as its name implies, is a blue protein present in human and many animal sera it is a glycoprotein with terminal sialic acid residues [8]. Caeruloplasmin was first isolated and characterized by... 

The final step in the denitrification process is carried out by the soluble enzyme nitrous oxide reductase (NoS). This enzyme has been isolated from a number of sources, and is unusual in a number of ways. In most cases, it is a homodimer of ca. 74 kDa subunits with ca. 4 Cu/subunit, but the enzyme is bright purple or pink as isolated, depending on conditions, and becomes the typical blue color expected for copper proteins only after reduction with dithionite. A variety of spectroscopic studies strongly suggest that the enzyme contains at least one mixed-valent, thiolate-bridged Cu(l)—Cu(ll) unit that may well be similar to a binuclear copper center in cytochrome c oxidase (23). The reaction catalyzed by the enzyme is deceptively simple ... 

The blue protein from A. faecalis strain S-6, which was isolated as a requirement for transferring electrons to a copper-containing nitrite reductase, has since been shown to have sequence homology with proteins arbitrarily designated pseudoazurin by Ambler and Tobari (1985), from Achromobacter cycloclastes and from Pseudomonas AMI. [Pseudomonas AMI also produces amicyanin, which is the recipient of electrons from methylamine dehydrogenase, (see below)]. In A. cycloclastes reduced pseudoazurin donates electrons to a copper nitrite reductase (Liu et ai, 1986), as it does in A. faecalis. Ambler and Tobari (1985)... 

Walker and Nicholas (338) have reported the isolation and 600-fold purification of an enzyme from P. aeruginosa, which reduces nitrite to nitric oxide. The preparation contained 1.5 nmoles of FAD per mg protein, a c-type cytochrome and an absorption band at 630-635 nm, suggestive of copper. As electron donors, reduced FMN, FAD, riboflavin, pyo-cyanine, and methylene blue were effective, but not NADH, NADPH, or reduced cytochrome c. The preparation required phosphate or sulfate for maximal activity. The cytochrome and the 630-635-nm band were reduced under anaerobic conditions with a suitable electron donor and readily oxidized by nitrite. The K for NaNOj is reported to be 3.1 X 10 ... 

Among the electron transport proteins, there are four known classes that have been isolated and studied by X-ray crystallography. These include cytochromes, iron-sulfur proteins, copper blue proteins, and... 

Quenching of excited-state [Ru(bipy)3] by reduced blue proteins involves electron transfer from the Cu with rate constants close to the diffusion limit for electron-transfer reactions in aqueous solution. It is suggested that the excited Ru complex binds close to the copper-histidine centre, and that outer-sphere electron transfer occurs from Cu through the imidazole groups to Ru. Estimated electron-transfer distances are about 3.3 A for plastocyanin and 3.8 A for azurin, suggesting that the hydrophobic bipy ligands of Ru " penetrate the residues that isolate the Cu-His unit from the solvent. ... 

The presence of ammonium ions in the alkaline copper increases the blank color, but decreases the blue-violet color produced from protein. This effect occurs at concentrations down to 0.05 M (S41), so that albumin in ammonium sulfate solutions must first be isolated before the quantitative biuret reaction can be applied. When present in higher concentrations, sodium sulfate and most other salts used for fractionating protein solutions intensify the biuret color (R27). However, the absorbance of the blank alkaline copper solutions containing no protein is also affected to the same extent, making it unnecessary to isolate the soluble protein before applying the biuret reaction. 

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