Journal of Molecular Biology
Regular articleThe structural role of the copper-coordinating and surface-exposed histidine residue in the blue copper protein azurin1
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
References (75)
- et al.
A crystallographic model for azurin at 3 Å resolution
J. Mol. Biol.
(1978) Structure of azurin from Alcaligenes denitrificans refinement at 1.8 Å resolution and comparison of the two crystallographically independent molecules
J. Mol. Biol.
(1988)- et al.
The role of His117 in the redox reactions of azurin from Pseudomonas aeruginosa
FEBS Letters
(1996) - et al.
The pH and redox-state dependence of the copper site in azurin from Pseudomonas aeruginosa as studied by EXAFS
Biochim. Biophys. Acta
(1986) - et al.
Crystal structure analyses of reduced (CuI) poplar plastocyanin at six pH values
J. Mol. Biol.
(1986) - et al.
Pulse sequences for removal of the effects of cross correlation between dipolar and chemical shift anistotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins
J. Magn. Reson.
(1992) ANSIGa program for the assignment of protein 1H 2D NMR spectra by interactive graphics
J. Magn. Reson.
(1989)- et al.
pH-dependent redox activity and fluxionality of the copper site in amicyanin from Thiobacillus versutus as studied by 300- and 600-MHz 1H NMR
J. Biol. Chem.
(1990) - et al.
EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobacillus versutus
Biochim. Biophys. Acta
(1991) - et al.
Axial ligand bonding in blue copper proteins
Inorg. Chim. Acta
(1992)
Backbone dynamics of Escherichia coli ribonuclease H1correlations with structure and function in an active enzyme
J. Mol. Biol.
Electron transfer in chemistry and biology
Biochim. Biophys. Acta
Crystal structure analysis of oxidized Pseudomonas aeruginosa azurin at pH 5.5 and pH 9.0. A pH-induced conformational transition involves a peptide bond flip
J. Mol. Biol.
Gradient-tailored water suppression for 1H-15N HSQC experiments optimized to retain full sensitivity
J. Magn. Reson. ser. A
A new method for parameterization of phase shift and backscattering amplitude
Physica ser. B
The crystal structures of reduced pseudoazurin from Alcaligenes faecalis S-6 at two pH values
FEBS Letters
Purification and characterization of a non-reconstitutable azurin, obtained by heterologous expression of the Pseudomonas aeruginosa azu gene in Escherichia coli
Biochim. Biophys. Acta
Raman spectroscopy as an indicator of Cu-S bond length in type 1 and type 2 copper cysteinate proteins
J. Am. Chem. Soc.
Cysteine ligand vibrations are responsible for the complex resonance Raman spectrum of azurin
J. Biol. Inorg. Chem.
X-ray absorption edge spectroscopy of copper(I) complexes. Coordination geometry of copper(I) in the reduced forms of copper proteins and their derivatives with carbon monoxide
Inorg. Chem.
Crystal structure of β-copper phthalocyanine
J. Chem. Soc.
Loop-directed mutagenesis of the blue copper protein amicyanin from Paracoccus versutus and its effect on the structure and the activity of the type-1 copper site
J. Am. Chem. Soc.
Structure and activity of type 1 Cu sites
Analysis of the backbone dynamics of interleukin-1 using two-dimentional inverse detected heteronuclear 15N-1H NMR spectroscopy
Biochemistry
Structure of metal site in Cd-substituted His117Gly mutant of azurin with and without addition of imidazole derivatives
Eur. J. Biochem.
Creation of type 1 and type 2 copper site by addition of exogenous ligands to the Pseudomonas aeruginosa azurin His117Gly mutant
J. Am. Chem. Soc.
Type I and type II copper sites obtained by external addition of ligands to a His117Gly azurin mutant
J. Am. Chem. Soc.
Resonance Raman spectroscopy of the azurin His117Gly mutant. Interconversion of type 1 and type 2 copper sites through exogenous ligands
Biochemistry
Electron transfer in ruthenium-modified plastocyanin
J. Am. Chem. Soc.
Structures of alumina-supported osmium clusters (HOs3(CO)10{OAl}) and complex (OsII(CO)n = 2or3{OAl}3) determined by extended X-ray absorption fine structure spectroscopy
J. Am. Chem. Soc.
The importance of not saturating H2O in protein NMR - application to sensitivity enhancement and NOE measurements
J. Am. Chem. Soc.
Crystal structures of modified apo-His117Gly and apo-His46Gly mutants of Pseudomonas aeruginosa azurin
J. Mol. Biol.
X-ray absorption spectroscopy of a new zinc site in the fur protein from Escherichia coli
Biochemistry
Copper - A ‘’modern’’ bioelement
Angew. Chem. Int. Ed.
Backbone dynamics of azurin in solutionthe slow conformational change associated with deprotonation of histidine 35
Biochemistry
Influence of preparation method on the metal cluster size of Pt/ZSM-5 catalysts as studied with extended X-ray absorption fine structure spectroscopy
J. Phys. Chem.
Purification and some properties of spinach plastocyanin
J. Biochem. (Tokyo)
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