Copper proteins EXAFS studies

Blue copper proteins, 36 323, 377-378, see also Azurin Plastocyanin active site protonations, 36 396-398 charge, 36 398-401 classification, 36 378-379 comparison with rubredoxin, 36 404 coordinated amino acid spacing, 36 399 cucumber basic protein, 36 390 electron transfer routes, 36 403-404 electron transport, 36 378 EXAFS studies, 36 390-391 functional role, 36 382-383 occurrence, 36 379-382 properties, 36 380 pseudoazurin, 36 389-390 reduction potentials, 36 393-396 self-exchange rate constants, 36 401-403 UV-VIS spectra, 36 391-393 Blue species... 

Some EXAFS data for copper proteins are given in Table 19.2. Confirming data from X-ray crystallography are also listed where known. Copper is particularly well suited for study by electron paramagnetic resonance. At the very simplest level, this... 

Intriguingly, the blue copper sites, especiaUy those with a carbonyl oxygen at the axial coordination position, display high affinity for Zn + ions. Mutants in which the Met is replaced by Gin or Glu preferentiaUy bind Zn + when expressed in heterologous systems, e.g., Escherichia coli. Examples include azurin, amicyanin, nitrite reductase, and possibly also plastocyanin (Diederix et al., 2000 Hibino et al., 1995 Murphy et al., 1995 Nar et al., 1992a Romero et al., 1993). In the case of azurin it has been shown that both wild-type and the Met—Gin mutant have the same affinity for both Zn +and Cu + (Romero ci a/., 1993). In addition, EXAFS studies showed that some preparations of blue copper proteins purihed from their natural sources also contain small fractions of Zn derivatives (DeBeer George, personal communication). 

The Copper Site. In a crystal form of ECAO shown to contain catalytically-active protein (Parsons et al., 1995), the eopper is penta-coordinated in approximate square pyramidal eonfiguration by four basal (equatorial) ligands (His 524, His 526, His 689 and a water [We]) and an apical (axial) water (Wa). The presence of equatorial and axial waters had been first reported by Barker et al. (1979) from EPR, water proton relaxation and kinetic studies on pig plasma amine oxidase and the prediction of histidines and waters as the copper ligands came from EXAFS studies by Scott and Dooley (1985). The equatorial water (We) is labile and not always present. In the HP AO structure (Li et al., 1998) it is present in some, but not all, of the six independent subunits in the same erystal. A comprehensive discussion of the spectroscopic properties of the copper site in amine oxidases, including the exchange rates for the equatorial and axial waters, is given in the review by Knowles and Dooley (1994). 

High-resolution crystallographic studies of type 1 copper proteins supplemented by EXAFS (extended X-ray absorption fine stmctme) data and analyses of sequence conservation show that the archetypal type 1 copper site has certain constant featmes. There exist several excellent reviews. 

Rusticyanin has a high reduction potential (680 mV), which is similar to that for the Type 1 Cu center in fungal as opposed to tree laccase (785 mV) (73). This trend is so far unexplained. From the sequence and EXAFS studies, His-Cys-His-Met coordination is a reasonable possibility for rusticyanin (55). It may well be that the reduction potential is determined by effects of a polypeptide backbone on Cu—S(Cys) and Cu—S(Met) bond distances and the Cu ligand field (74). If this is the case, however, rusticyanin would be expected to have one or both Cu—S distances shorter than in other blue copper proteins, which is not borne out by information from EXAFS (Table IV). A further possibility that the Cu(I) form is three-coordinate, as in the case of plasto-cyanin at low pH (Fig. 2), has no strong support at present (75). 

Our biomimetic investigations have focused on the metalloproteins hemocyanin (He) (11-17) and tyrosinase (11,12,14,16,18,29), which contain two copper ions in their active center. The function of hemocyanin is to bind and transport dioxygen in the hemolymph of molluscs and arthropods. Studies employing EXAFS spectroscopy have shown that in the deoxy form, two (19-21) or three (13,21) imidazole units fiom protein histidine residues coordinate to each cuprous ion. Upon addition of O2 to give oxy-Hc, considerable changes take place in the coordination sphere giving rise to tetragonally coordinated Cu(II) ions... 

Metallothioneins are a unique and widely distributed group of proteins. They are characterized by their low molecular weight (—6000), high cysteinyl content, and the ability to bind substantial numbers of metal ions (43). The proteins bind copper and zinc, thereby providing a mobile pool as part of the normal metabolism of these elements, and offer protection from the invasion of inorganic forms of the toxic elements cadmium, lead, and mercury. In addition, other metals, such as iron and cobalt, can be induced to bind. XAS is ideally suited to probe the environment of these different metal atoms (see Fig. 1), and the structural interpretations obtained from an analysis of the EXAFS data obtained in several such studies are summarized in Table 1(44). Thus, in each case, the data are consistent with the primary coordination of the metal deriving from the cysteinyl residues. 

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