Azurin ligands

In the blue, Type I copper proteins plastocyanin and azurin, the active-site structure comprises the trigonal array [CuN2S] of two histidine ligands and one cysteine ligand about the copper,... 

Antholine, W.E., Hanna, P.M., and McMillan, D.R. 1993. Low frequency EPR of Pseudomonas aeruginosa azurin analysis of ligand superhyperfine structure from a type 1 copper site. Biophysical Journal 64 267-272. 

Figure 5.9 Ligands (A) N,S-mpy, (B) N4-mpy, (C) N2S2-mpy, and (D) N2S2-inim designed to mimic azurin and plastocyanin active sites. (Adapted with permission of The Royal Society of Chemistry from Malachowski, M. R. Adams, M. Elia, N. Rheingold, A. L. Kelly, R. S. J. Chem. Soc., Dalton Trans., 1999, 2177-2182.

Simple thermodynamic considerations state that the reduction process is favoured (i.e. more positive cu(ii)/cu(p potential values are obtained) if the electron transfer is exothermic (AH° negative) and if the molecular disorder increases (AS° positive). It is therefore evident that the positive potential value for the reduction of azurin (as well as that of the most blue copper proteins) is favoured by the enthalpic factor. This means that the metal-to-ligand interactions inside the first coordination sphere (which favour the stability of the reduced form over the oxidized form) prevail over the metal complex-to-solvent interactions inside the second... 

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]. 

When copper is bound to one sulfur atom of a cysteine and two nitrogens of two histidines in an essentially tetrahedrally distorted - trigonal ligand environment (type I copper proteins), the excited levels are low in energy, and the values are reduced to about 5 x 10 ° s (29). Examples are blue copper proteins, like ceruloplasmin and azurin, and copper(II) substituted liver alcohol dehydrogenase (30-32). 

As an example on the relationship between proton relaxivity, electron relaxation and coordination environment, we report the case of azurin and its mutants. The relaxivity of wild type azurin is very low (Fig. 6) due to a solvent-protected copper site, the closest water being found at a distance of more than 5 A from the copper ion. The fit, performed with the Florence NMRD program, able to take into account the presence of hyperfine coupling with the metal nucleus (Ay = 62 x 0 cm , see Section II.B) indicates Tie values of 8 X 10 s. Although the metal site in azurin is relatively inaccessible, several mutations of the copper ligands open it up to the solvent. The H NMRD profiles indicate the presence of water coordination for the... 

It has long been known that, under some conditions at least, electron transfer between the c and d hemes of the P aeruginosa enzyme is slow and requires times of the order of seconds (22). What does this mean It is not necessarily related to the loss of the hydroxide ligand from the d heme iron, because under some experimental conditions used, azurin (a cupredoxin) was present and the enzyme was reduced at the outset,... 

Compound I will accept a further electron from azurin and decays to a species known as compound II with a bimolecular rate constant of 6 X 10 M s (84). It is anticipated that compound II is a form of CCP containing two ferric hemes, but possibly not identical to the structurally defined fully oxidized enzyme as isolated. This is because during turnover at room temperature there is no obvious reason for the histidine ligand displaced from the peroxidatic heme iron to return. Consequently, it might be assumed that compound II is structurally related to the MV conformer rather than the resting enzyme. 

Fig. 2. Copper site in azurin. In this and subsequent figures the following conventions have been used, (a) The copper site is generally an enlargement of (b). The copper site is a dotted sphere, the ligand residues are represented by bonds connecting atoms in the side chain, and, where possible, the atoms bonded to the copper atom are identified by atom type. Ribbons represent portions of the backbone structure near the copper. In (b) of each figure, the main-chain polypeptide is represented by a ribbon fit to the main-chain coordinates, and the amino and carboxy termini are indicated by N and C, respectively, (c) A schematic version drawn from (b). Solid arrows represent main-chain regions participating in the /3 sheet roughly above the plane of the paper, while dotted or light arrows are the...

In contrast to azurin, the plant plastocyanins have a conserved negative patch of residues adjacent to a putative redox partner-binding site. Plastocyanin has, in addition, the hydrophobic face into which the edge of the second histidine ligand is inserted. 

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