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The structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin1

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Abstract

Copper K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy and 15N NMR relaxation studies were performed on samples of a variant azurin in which the surface-exposed histidine ligand of the copper atom (His117) has been replaced by glycine. The experiments were performed to probe the structure of the active site and the protein dynamics. The cavity in the protein structure created by the His → Gly replacement could be filled by external ligands, which can either restore the spectroscopic properties of the original type-1 copper site or create a new type-2 copper site. The binding of external ligands occurs only when the copper atom is in its oxidised state. In the reduced form, the binding is abolished. From the EXAFS experiments, it is concluded that for the oxidised type-1 copper sites the protein plus external ligand (L) provide an NSS∗L donor set deriving from His46, Cys112, Met121 and the external ligand. The type-2 copper site features an S(N/O)3 donor set in which the S-donor derives from Cys112, one N-donor from His46 and the remaining two N or O donors from one or more external ligands. Upon reduction of the type-1 as well as the type-2 site, the external ligand drops out of the copper site and the coordination reduces to 3-fold with an SS∗N donor set deriving from His46, Cys112 and Met121. The Cu-Sδ(Met) distance is reduced from about 3.2 to 2.3 Å. Analysis of the NMR data shows that the hydrophobic patch around His117 has gained fluxionality when compared to wild-type azurin, which may explain why the His117Gly variant is able to accommodate a variety of external ligands of different sizes and with different chelating properties. On the other hand, the structure and dynamics of the β-sandwich, which comprises the main body of the protein, is only slightly affected by the mutation. The unusually high reduction potential of the His117Gly azurin is discussed in light of the present results.

Introduction

Blue copper proteins, like azurin, belong to a class of mononuclear copper proteins that contain a so-called type-1 copper site (or blue copper site). These proteins are relatively small (8–14 kDa) and function in electron transport. For azurin, 3D structures have been solved for the proteins in two oxidation states and at different pH values Adman et al 1978, Shepard et al 1990, Baker 1988. Besides a small conformational change around residues 35–37, which is due to protonation/deprotonation of histidine 35, the overall protein structures are similar. The copper ion is coordinated by the Sγ of a cysteine residue and Nδ of two histidine residues (Figure 1). This strong N2S donor set is conserved in all members of the blue copper protein family. The highly covalent nature of the copper-sulphur bond gives blue copper proteins their unique spectroscopic properties. These include an intense absorption near 600 nm (ε = 3000–6000 M−1 cm−1) arising from a (Cys)S → Cu(II) charge transfer and small hyperfine splitting (<100 × 10−4 cm−1) in the g region of their electron paramagnetic resonance (EPR) spectra (for a review, see Solomon et al., 1992). For the copper site of azurin, a less conserved ligand, the Sδ of a methionine residue serves as an axial ligand, resulting in a trigonal pyramidal coordination geometry around the copper. A second axial group, the carbonyl oxygen atom of a glycine residue, has an electrostatic interaction with the copper ion (Lowery & Solomon, 1992) and is unique for azurins. Azurins are unique also in that they contain a disulphide bond between Cys3 and Cys26, which connects the first two N-terminal β-strands in the structure.

Structures of reduced azurin Shepard et al 1990, Groeneveld et al 1986 are nearly identical with those of the oxidised protein, which means that the reorganisation energy for reduction or oxidation is low. Low reorganisation energies facilitate electron transfer under biological conditions, i.e. at low driving forces (Marcus & Sutin, 1985).

In contrast, conformational changes are observed at neutral to low pH in the reduced blue copper proteins amicyanin, plastocyanin and pseudoazurin. The bond between the reduced copper ion (Cu(I)) and the surface-exposed histidine residue is lost at low pH due to protonation of Nδ of this histidine residue. X-ray diffraction studies on single crystals of reduced plastocyanin and pseudoazurin at different pH values show that upon protonation of this histidine residue the copper ion moves into the plane of the remaining ligands (Sγ(Cys), Nδ(His) and Sδ(Met)) Guss et al 1986, Vakoufari et al 1994. Extended X-ray absorption fine structure (EXAFS) studies on amicyanin lead to a similar picture (Lommen et al., 1991). In this conformation, the blue copper sites exhibit high midpoint potentials, while oxidation rates decrease appreciably Katok et al 1962, Lommen and Canters 1990, DiBilio et al 1998, Sykes 1985, Segal and Sykes 1978. Therefore, this state is sometimes said to be “redox inactive”.

In this respect, azurin is different: the homologous His117 does not protonate when azurin is in its oxidised or reduced form. However, the disappearance of His117 from the coordination sphere of the copper ion could be mimicked by site-directed mutagenesis. The azurin mutant in which this surface-exposed histidine residue has been removed (His117Gly azurin) was shown to parallel the redox properties of the blue copper proteins in which the surface-exposed histidine residue protonates (unpublished results). Interestingly, the copper site of this mutant in addition appears accessible to exogenous ligands Andrew et al 1997, Danielsen et al 1995, den Blaauwen et al 1991, den Blaauwen et al 1993, den Blaauwen and Canters 1993, Gorren et al 1996, van Pouderoyen et al 1996. Depending on the kind of ligand, different types of copper site have been obtained: some ligands, like Cl, Br, imidazole (derivatives) and pyridine (derivatives), when added to the oxidised form of His117Gly azurin, restore the spectroscopic features that are characteristic of type-1 copper sites. Other ligands, like water, histidine and histamine, create novel copper sites with spectroscopic features that are more similar to “normal” or type-2 copper sites Kaim and Rall 1996, Solomon et al 1992.

The electron transfer (ET) properties of these type-1 and type-2 sites in His117Gly azurin are different. For instance, intramolecular ET from the disulphide bridge to these copper sites was investigated with pulse radiolysis (O. Farver et al., unpublished results) and it was found that His117Gly azurin in the presence of imidazole exhibits ET that is faster than in wild-type azurin. However, when the imidazole was replaced by water, the ET rate was much lower than for wild-type azurin, indicating either an increase in reorganisation energy or a decrease of the covalent character of the bond between the (Cys)Sγ and the copper ion.

Although a range of external ligands exhibit good affinity for the copper ion in oxidised His117Gly azurin, electrochemical studies showed that the affinity for the reduced form was either absent (Cl) or extremely low (imidazole) (unpublished results). By analogy with the low-pH form of other reduced blue copper proteins (amicyanin, plastocyanin and pseudoazurin), it was proposed that the Cu(I) ion in His117Gly azurin may move into the plane of the remaining ligands, Sγ of Cys112, Nδ of His46 and Sδ of Met121.

In order to better understand the intriguing properties of His117Gly azurin, it is necessary to obtain further structural and dynamic information about His117Gly azurin in the presence of different ligands. Previously, the crystal structure of His117Gly azurin was solved, but it was found that during crystallisation Cys112 had become oxidised and the copper ion was lost (Hammann et al., 1997). Therefore, this structure did not provide us with information about the native form of the His117Gly variant. Also a study of the dynamics of His117Gly azurin is relevant. The replacement of His117 by a Gly results in two adjacent glycine residues (at 116 and 117) in the loop connecting the last two C-terminal β-strands. Together with the loss of the coordination of the His117 Nδ to the copper ion, this may increase the overall mobility of this loop and thus the possibility to accommodate larger ligands. To obtain further information on the structure and the dynamics of the copper site of the His117Gly azurin variant, K-shell copper EXAFS and 15N NMR studies were performed on His117Gly azurin in solution. From the EXAFS spectra, information is acquired about the direct surroundings (2–3 Å) of the copper in both the reduced and the oxidised state. The 15N NMR data supply information about the overall fold of reduced His117Gly azurin and on the mobility of the C-terminal loop on which residue 117 is located.

Section snippets

EXAFS spectra

EXAFS spectra were recorded on two wild-type and eight His117Gly azurin samples (Figure 2) with S/N ratios comparable to data from the literature Lommen et al 1991, Murphy et al 1993, Strange et al 1995, Strange et al 1996, Jacquamet et al 1998. The results can be divided into three groups, depending on the features in the raw and FT EXAFS spectra (Figure 2).

Wild-type azurin

In the EXAFS study on oxidised wild-type azurin, a single sulphur atom at 2.15 Å from the copper ion is observed, which is 0.09 Å less than the distance previously reported on the basis of X-ray structures at 1.8 Å resolution (Nar et al., 1991). EXAFS studies, as well as two crystal structures at 1.8 Å resolution, of azurin from Alcaligenes denitrificans showed distances of 2.19 Å (Groeneveld et al., 1986), 2.12 Å (Tullius et al., 1978), 2.12 Å (Murphy et al., 1993), 2.12 Å and 2.17 Å (Baker,

Conclusion

When His117Gly azurin was characterised for the first time it appeared impossible to convert the reduced protein back to its oxidised form den Blaauwen et al 1991, den Blaauwen and Canters 1993 and it was thought that the active site had been damaged during the reduction or isolation process. Later, crystallographic research on what subsequently turned out to be slightly damaged protein (Hammann et al., 1997), confirmed that the Sγ of the copper ligand Cys112 could easily be oxidised. Yet, the

EXAFS samples

Wild-type azurin and His117Gly azurin were expressed and purified as described van de Kamp et al 1990a, den Blaauwen and Canters 1993.

Using ultra-filtration, oxidised wild-type azurin was concentrated to ∼10 mM (concentration determined from the absorption at 626 nm (ε = 5700 M−1 cm−1) (van de Kamp et al., 1990b)) in 20 mM Mes (pH 6.8). Reduction was achieved by adding a small excess of sodium dithionite/sodium hydrogen carbonate (1:2, w/w) after flushing the sample extensively with argon. To

Acknowledgements

We thank Professor D.C. Koningsberger for his interest in this work. G.W.C. thanks Dr E.I. Solomon for an illuminating discussion on the EXAFS of Cu model compounds. We gratefully acknowledge the financial support of the Ministry of Economic Affairs, the Ministry of Education, Culture and Science and the Ministry of Agriculture, Nature Management and Fishery in the framework of an industrial relevant research programme of the Netherlands Association of Biotechnology Centres in the Netherlands

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