Azurins spectra

Differences between the spectra of fluorescence and phosphorescence are immediately obvious. For all tryptophans in proteins the phosphorescence spectrum, even at room temperature, is structured, while the fluorescence emission is not. (Even at low temperatures the fluorescence emission spectrum is usually not structured. The notable exceptions include a-amylase and aldolase, 26 protease, azurin 27,28 and ribonuclease 7, staphylococcal endonuclease, elastase, tobacco mosaic virus coat protein, and Drosophila alcohol dehydrogenase 12. )... 

Mavicyanin (Mj = 18,000) is obtained from green squash (Cucurbito pepo medullosa), where it occurs alongside ascorbate oxidase [64]. It has a peak at 600 nm (e 5000 M cm and reduction potential of 285 mV. Further studies on this and the mung bean and rice bran proteins [65, 66] would be of interest. All the above type 1 Cu proteins have an intense blue color and characteristic narrow hyperfine EPR spectrum for the Cu(II) state. Table 3 summarizes the properties of those most studied. There is some variation in reduction potential and position of the main visible absorbance peak. In the case of azurin, for example, the latter is shifted from 597 to 625 nm. Stellacyanin has no methionine and the identity of the fourth ligand is therefore different [75]. The possibility that this is the 0(amide) of Gln97 has been suggested [63b]. It now seems unlikely that the disulfide is involved in coordination. Stellacyanin has 107 amino acids, with carbohydrate attached at three points giving a 40% contribution to the M, of 20,000 [75]. 

Unfortunately, bond lengths have not been reported for the copper center of Cbp. Its spectrum (like that of plastocyanin) is much more like that of the A. faecalis pseudoazurin than azurin. Since little variability of the Cu-Sy bond has been seen in the three structures described above, and since the major difference between pseudoazurin and plastocyanin (or azurin) is the length of the Cu-Met S8 bond, this would suggest that the Cu-Met bond is short in this protein, as well. Its EPR is also rhombic, again, like that of pseudoazurin. 

A recently characterized single-domain copper protein, auracyanin (Trost et al., 1988), is a dimeric protein which has a visible spectrum more like that of the A. faecalis cupredoxin (pseudoazurin, subgroup II see Table II) than that of either azurin or plastocyanin, but, because of its cysteine content and rhombic EPR, it has been put in the other class in Table II. 

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... 

Similarities with respect to the location of cysteine, histidine and methionine residues in the proteins of azurin, plastocyanin and ceruloplasmin indicate that the type 1 centres in ceruloplasmin are similar to those in the other two proteins. The nine-line superhyperfine splitting in the ESR spectrum of the type 2 Cu has been interpreted in terms of four equivalent nitrogen ligands.978 This was observed in a protein from which the type 1 copper was depleted by dialysis against ascorbate. 

Fig. 5.35. ID H NMR spectrum (200 MHz, 298 K) of Co(II)-azurin in H2O (adapted from [96]). A schematic drawing of the metal site in Pseudomonas aeruginosa native azurin is shown in the upper left comer.

Fig. 5.49. 300 MHz H NMR spectrum of nickel(II)-substituted azurin at pH 7.0 and 303 K, and a schematic drawing of the metal coordination polyhedron (adapted from [97]).

Figure 1. Reduced-minus-oxidized difference absorption spectrum of Ps. aeruginosa azurin. Sample and reference cell contained 6.8 X 10 5 M solutions of reduced and oxidized protein, respectively. Hydrogen, with platinum black as catalyst, was used as reductant (1). Medium ...

The additional effects in the aromatic region of the difference spectrum (250-300 nm) are probably caused by aromatic transitions which are influenced by the redox state of the copper. The shoulder at 270 nm, which occurs in all three proteins, could result from an increase in tyrosine absorption. In this context, it is interesting to recall that Tyr 108 (azurin numbering), which is relatively close to the proposed copper ligands Cys 112 and Met 121, is completely invariant both in azurin and plastocyanin and may therefore be an obligatory constituent of the copper site. 

Fig.4. NMR spectra of cobalt(II)-substituted (A) Pseudomonas aeruginosa azurin (Moratal Mascarell a/., 1993b) ( ) Achromohacter cycloclastes pseudoazurin (Fernandez et al., submitted for publication), and (C) Rhus vernacifera stellacyanin (Vila, 1994). Spectra (A) and (C) were recorded at 200 MHz at 313 K, whereas spectrum (B) was recorded at 600 MHz and 318 K. All the samples were in 50 mM phosphate buffer at pH 6 in water solution.

Figure 17 Low-temperature (77 K) RR spectrum of P. aeruginosa azurin obtained with 647.1-mn excitation wavelength and structural drawing of the active site showing distances (A) to the three strong (Cysl 12, His46, Hisll7) and two weak ligands (Metl21, Gly45)...

Figure 23 Changes in the UV-visible absorption spectrum of P. aeruginosa M121G azurin on sequential addition of aqueous sodium azide...

The excitation wavelength-dependent RR spectra (Figme 28) also provided unambiguous evidence for the nature of the CT electronic transition at 440 nm as due to (Cys)S — Ni(ll). Figure 31 compares the Ni(ll)-azurin optical absorption spectrum with excitation profiles for the three most intense M-sensitive RR bands at 346,360, and 390 cm . The profiles are quite similar and closely track the absorption band in the violet region. 

Figure 30 Effects of Ni-isotope substitution on the 200-430 cm" RR spectrum of P. aeruginosa azurin Italic numbers show the [v( Ni) — v( Ni)] band shifts...

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