Blue proteins

Copper Blue Proteins.— The present understanding of the chemistry of the copper blue electron-transfer proteins has taken a significant step forward with the publication of molecular structures for azurin and plastocyanin.  

Together with EXAFS, resonance Raman, metal-ion 

Cross reactions of plastocyanins and azurins from various sources with cytochromes can be fitted to the Marcus equation using constant self-exchange rate constants of 6.6 x 10 s for the plastocyanins, 9.9 x 10 s for Ps. 

A mechanism involving protein-complex association prior to electron transfer has been proposed to explain deviations from first-order behaviour in the oxidations and reductions of parsley and spinach plastocyanins and Ps. aeruginosa azurin with inorganic reagents. The oxidations of reduced plastocyanin by [Co(phen)3] + and [Fe(CN)i ] have a pH dependence indicative of redox-inactive protonated forms of the protein with pKa values 6.1 and 5.7 respectively. For the reaction with [Co(phen)3] +, 

Similar results are noted for azurin where, for the reduced protein ACu, different binding sites witlj pATa values 7.6 ([Co(phen)3] +), 7.1 ([Fe-(CN)e] ), and no pH dependence ([Co(4,7-DPSphen)3] ) are reported. No dramatic switch-off in reactivity, as with plastocyanin, is noted, however, and the trend in the rates with protonation reflects that expected for the effect of electrostatic attraction on substrate binding. The pATa of 7.1 for [Fe(CN)8] reaction is shifted to pATa 6.1 for [Fe(CN)6] reduction of ACu and, by comparing the kinetic pATa values with those obtained by n.m.r., tentative assignments to histidines-35 and -83, which are not bound to the copper ion, are made. 

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Nakamura K, Go N. 2005. Function and molecular evolution of multicopper blue proteins. Cellular Mol Life Sci 62 2050-2066. 

Rossi, F. M. V., Blakely, B. T., and Blau, H. M. (2000). Interaction blues protein interactions monitored in live mammalian cells by fl-galactosidase complementation. Trends Cell Biol. 10, 119-122. 

Edgar PF Comparative proteome analysis. Tissue homogenate from normal human hippocampus subjected to two-dimensional gel electrophoresis and Coo-massie blue protein staining. Mol Psychiatry 2000 5 85-90. 

Type I copper enzymes are called blue proteins because of their intense absorbance (s 3000 M-1 cm- ) in the electronic absorption spectrum around... 

Let us start our examination with the prototypical blue protein plastocyanin, found in the thylacoid membrane of chloroplasts, where it acts as an electron carrier in photosynthesis (see Figure 1). As Figure 30 illustrates, the active site of plastocyanin is formed of a Cu(II) ion (pseudo)tetrahedrally coordinated to two histidine nitrogen atoms and... 

The molecular structure of another blue protein, the phytocyanin (phytocyanins are electron carriers found in the non-photosynthetic part of plants) cucumber basic protein (FW=10 100), also known as plantacyanin, is shown in Figure 33.60... 

As illustrated in Figure 35, the same redox behaviour is exhibited by the positively charged blue protein pseudoazurin (FW=13 400 p/ = 8.9), a class of proteins devoted to reduction of NCV to NO in denitrifying bacteria. 

Another interesting blue protein is stellacyanin (FW = 20 000) from the Japanese lacquer tree Rhus vernicifera, in which, with respect to the other cupredoxins, glutamine replaces the methionine ligand.64 Stellacyanin also bears an overall positive charge (p/=9.9). It, therefore, gives a reversible Cu(II)/Cu(I) response at a glassy carbon electrode in aqueous solution (pH 7.6).61 The formal electrode potential of the Cu(II)/Cu(I) reduction (E01 = + 0.18 V vs. NHE) is the lowest among cupredoxins. 

Finally, we examine azurin, a blue protein (FW = 14 000) devoted to bacterial electron transport, the copper centre of which has a penta-coordinate trigonal bipyramidal geometry, at variance with all the other cupredoxins, Figure 39.73... 

The crystal structure of the pseudoazurin from Alcaligenes faecalis S-6 sometimes referred to as the blue protein (also as cupredoxin), has been reported to 2.0 A [74]. The protein folds in /3-sandwich which is described as being similar to plastocyanin and azurin. 

There are a number of excellent sources of information on copper proteins notable among them is the three-volume series Copper Proteins and Copper Enzymes (Lontie, 1984). A review of the state of structural knowledge in 1985 (Adman, 1985) included only the small blue copper proteins. A brief review of extended X-ray absorption fine structure (EXAFS) work on some of these proteins appeared in 1987 (Hasnain and Garner, 1987). A number of new structures have been solved by X-ray diffraction, and the structures of azurin and plastocyanin have been extended to higher resolution. The new structures include two additional type I proteins (pseudoazurin and cucumber basic blue protein), the type III copper protein hemocyanin, and the multi-copper blue oxidase ascorbate oxidase. Results are now available on a copper-containing nitrite reductase and galactose oxidase. 

Cucumber basic blue protein has been put in a class of its own in view of having a disulfide, unUke plastocyanin, although its fold is generally like that of plastocyanin. 

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)... 

The salient features of A. faecalis pseudoazurin are that (1) it has a Cu-Met bond length shorter than that of either plastocyanin or azurin (see Table III) (2) it has only one NH - S bond, as does plastocyanin and (3) its overall architecture resembles plastocyanin (see Fig. 4), with an extended carboxy terminus folded into two a helices [a preliminary sequence comparison suggested that the folding would resemble plastocyanin (Adman, 1985)]. It retains the exposed hydrophobic face found in azurin and plastocyanin. Just how it interacts with nitrite reductase is still a subject of investigation. It is intriguing that the carboxy-terminal portion folds up onto the face of the molecule where the unique portions of other blue proteins are the flap in azurin, and, as we see below in the multi-copper oxidase, entire domains. 

The gene for this protein has also been isolated from A. faecalis and sequenced and expressed in Escherichia coli cells (Yamamoto et al., 1987). Like azurin, it contains an amino-terminal signal sequence, suggesting that it is secreted into the periplasm in A. faecalis, although not all of the blue protein is found in this fraction. Mutants of this protein are being made and characterized in T. Beppu s laboratory (Univ. of Tokyo) (personal communication, 1989). 

Cucumber basic blue protein (Cbp) is a protein without known function, also known as cusacyanin or plantacyanin. Its structure (Guss et al., 1988) completes the repertoire of cupredoxins with known structures. The topology of its folding is similar (Fig. 5) to those of plastocyanin and azurin, as might have been expected from sequence similarities and... 

Fig. 5. (a) Copper site in cucumber basic blue protein (Cbp). (b) Ribbon drawing of the Cbp backbone, (c and d) Schematic of Cbp topology. 

Although crystals have been reported for two amicyanins (Petratos et al., 1988b Lim et al., 1986), the type 1 blue protein, which is an electron acceptor for methylamine dehydrogenase (Tobari and Harada, 1981 van Houweligen et al., 1989), neither study has yet been completed. The structure of methylamine dehydrogenase from Thiobacillus versutus (not a copper protein) has recently been reported (Vellieux et al., 1989). The amicyanin from P. denitrificans has actually been cocrystallized with methylamine dehydrogenase (F. S. Mathews, personal communication. 

When the complete amino acid sequence of ceruloplasmin was determined, an internal threefold repeat suggested gene triplication (Ta-kahashi et al., 1983). Moreover, sequence similarity to the small blue copper domains suggested that there were at least two domains with blue copper-binding sites. Analysis of fragments of laccase sequence indicated that there might be a relationship of this to small blue proteins and ceruloplasmin as well (Ryden, 1988). 

Fig. 5 2D PAGE of CSF before and after depletion with Cibacron blue/protein G affinity resins...

Having elucidated, in combination with X-ray structural data, the characteristics of the copper site coordination in blue proteins in extenso, the challenge for EPR spectroscopy (and other techniques) is now to find ways to model the electron transfer (ET) in a realistic fashion. At present EPR is, however, mostly used to ascertain that the coordination of copper in the experimental ET chain models employed is not disturbed prior to ET. Plastocyanin is the electron carrier in photosynthesis. Indications of structural origins of impaired ET in... 

The blue color of these "type 1" copper proteins is much more intense than are the well known colors of the hydrated ion Cu(H20)42+ or of the more strongly absorbing Cu(NH3)42+. The blue color of these simple complexes arises from a transition of an electron from one d orbital to another within the copper atom. The absorption is somewhat more intense in copper peptide chelates of the type shown in Eq. 6-85. However, the -600 nm absorption bands of the blue proteins are an order of magnitude more intense, as is illustrated by the absorption spectrum of azurin (Fig. 23-8). The intense blue is thought to arise as a result of transfer of electronic charge from the cysteine thiolate to the Cu2+ ion.520 521... 

In addition to its previously mentioned role in copper transport, ceruloplasmin is an amine oxidase, a superoxide dismutase, and a ferrooxidase able to catalyze the oxidation of Fe2+ to Fe3+. Ceruloplasmin contains three consecutive homologous 350-residue sequences which may have originated from an ancestral copper oxidase gene. Like ascorbate oxidase, this blue protein contains copper of the three different types. Blood clotting factors V and VIII (Fig. 12-17), and the iron uptake protein Fet3 (Section A,l) are also closely related. 

Gaussian curves (normal distribution functions) can sometimes be used to describe the shape of the overall envelope of the many vibrationally induced subbands that make up one electronic absorption band, e.g., for the absorption spectrum of the copper-containing blue protein of Pseudomonas (Fig. 23-8) Gaussian bands are appropriate. They permit resolution of the spectrum into components representing individual electronic transitions. Each transition is described by a peak position, height (molar extinction coefficient), and width (as measured at the halfheight, in cm-1). However, most absorption bands of organic compounds are not symmetric but are skewed... 

Figure 23-8 Resolution of the visible circular dichroism (ellipticity) spectrum (A) and absorption spectrum (B) of the Pseudomonas blue protein into series of overlapping Gaussian hands (—). The numbers 1 to 6 refer to hands of identical position and width in both spectra. Absorption envelopes resulting from the sum of the set of overlapping Gaussian bands (—) correspond within the error of the measurement to the experimental spectra. The dashed part of the CD envelope above 700 nm was completed by a curve fitter with the use of a band in the position of hand 1 of the absorption spectrum. From Tang et al.68...

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