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Acta Crystallographica Section F: Structural Biology Communications logoLink to Acta Crystallographica Section F: Structural Biology Communications
. 2016 Jun 22;72(Pt 7):558–563. doi: 10.1107/S2053230X16009237

Exogenous acetate ion reaches the type II copper centre in CueO through the water-excretion channel and potentially affects the enzymatic activity

Hirofumi Komori a, Kunishige Kataoka b, Sakiko Tanaka b, Nana Matsuda b, Yoshiki Higuchi c, Takeshi Sakurai b,*
PMCID: PMC4933006  PMID: 27380373

The acetate-bound form of copper was found in the crystal structure of the multicopper oxidase CueO. The exogenous acetate ion can reach the copper active site through the water channel in the CueO molecule.

Keywords: bioinorganic chemistry, enzyme catalysis, metalloproteins, CueO, multicopper oxidase

Abstract

The acetate-bound form of the type II copper was found in the X-ray structure of the multicopper oxidase CueO crystallized in acetate buffer in addition to the conventional OH−-bound form as the major resting form. The acetate ion was retained bound to the type II copper even after prolonged exposure of a CueO crystal to X-ray radiation, which led to the stepwise reduction of the Cu centres. However, in this study, when CueO was crystallized in citrate buffer the OH−-bound form was present exclusively. This fact shows that an exogenous acetate ion reaches the type II Cu centre through the water channel constructed between domains 1 and 3 in the CueO molecule. It was also found that the enzymatic activity of CueO is enhanced in the presence of acetate ions in the solvent water.

1. Introduction  

Multicopper oxidase (MCO) belongs to a class of copper enzymes that harbour four Cu atoms classified as type I copper (T1Cu), type II copper (T2Cu) and type III copper (T3Cu) (Komori & Higuchi, 2010 ▸, 2015 ▸; Sakurai & Kataoka, 2011 ▸). MCOs oxidize a variety of substrates such as phenols, aromatic amines and transition-metal ions at T1Cu, and reduce O2 at the trinuclear copper centre (TNC), which is comprised of one T2Cu atom and a pair of T3Cu atoms. During the four-electron reduction of O2 to H2O by MCOs, activated oxygen species are not formed or are not released from the TNC if they are formed. Therefore, MCOs have been used in biofuel cells as a cathodic enzyme in addition to uses in clinical tests and for pigment formation (Miura et al., 2009 ▸).

In the resting form of MCOs, T1Cu and T2Cu can be detected by electron paramagnetic resonance (EPR) spectroscopy, but T3Cu atoms are not detected owing to antiferromagnetic interaction between them. This classical resting form of the TNC, in which the T3Cu atoms are bridged by only an OH− ion, was first observed in the crystal structure of ascorbate oxidase and has subsequently been found in other MCOs (Messerschmidt et al., 1989 ▸). However, variants with an O-centred TNC structure as an intermediate in the O2-reduction process (Huang et al., 1999 ▸; Komori et al., 2012 ▸; Morishita et al., 2014 ▸) and partly reduced TNC structures have recently been discovered in resting MCOs and mutants (Polyakov et al., 2009 ▸). Further, an exogenous Cl−-binding TNC has also been reported, presumably because crystallization had been performed in the presence of this small anion (Hakulinen et al., 2002 ▸). The binding of exogenously soaked azide and peroxide to the TNC have also been reported for ascorbate oxidase, although discussion of the O2-reduction mechanism has become more complicated owing to their diverse binding modes (Messerschmidt et al., 1993 ▸).

As a result of studies on plant laccase (Huang et al., 1999 ▸; Zoppellaro et al., 2001 ▸), CueO (Kataoka et al., 2009 ▸), bilirubin oxidase (Kataoka et al., 2005 ▸), Fet3p (Augustine et al., 2007 ▸) and CotA (Roberts et al., 2002 ▸; Mizutani et al., 2010 ▸; Taylor et al., 2005 ▸; Enguita et al., 2003 ▸), it has been revealed that acidic amino acids play a vital role in the transport of H+ and the tuning of the redox potential of copper centres by forming a hydrogen bond to the ligand groups and/or specific water molecules. Glu506 involved in the hydrogen-bond network leading from solvent water to the OH− ion bridged between the T3Cu atoms in CueO (greenish-blue Cα atom and side chain shown as a ball-and-stick model and the greenish-blue tunnel in Fig. 1 ▸) is involved in the transport of H+ to O2 (Iwaki et al., 2010 ▸; Komori et al., 2013 ▸). On the other hand, Asp112 located in the channel constructed between domain 1 and domain 3 in the CueO molecule (purple Cα atom and side chain shown as a ball-and-stick model and the purple tunnel in Fig. 1 ▸) has been considered to be involved in the activation of O2 and the excretion of H2O (Ueki et al., 2006 ▸; Kataoka et al., 2009 ▸).

Figure 1.

Figure 1

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Tunnels leading from the protein surface to the TNC in CueO. The greenish-blue tunnel is the H+-transport channel, in which specific water molecules are present. At the end of the hydrogen-bond network the side chain and Cα of Glu506 (greenish-blue ball-and-stick representation) are located to form a hydrogen bond to the OH– bridged between the T3Cu atoms via a water molecule. The purple tunnel constructed between domain 1 and domain 3 is for the excretion of water. Near its end, the side chain and Cα of Asp112 (purple ball-and-stick representation) are located. The role of the grey tunnel is not known, but it might be involved in the introduction of O2. The red arrows indicate the directions of flow of H+, O2 and H2O via the greenish-blue, grey and purple tunnels, respectively, and of e− from T1Cu to the TNC via the His–Cys–His triad that connects them. Blue, green and red spheres are the T1Cu, T2Cu and T3Cu atoms, respectively. Tunnels are depicted using Caver Analyst (Kozlikova et al., 2014 ▸) with the previously published CueO coordinates (PDB entry 4e9s).

In a series of crystal structure analyses of CueO under low to high X-ray dose conditions (Komori et al., 2014 ▸), we showed changes in the structure of the TNC from data set 1 to data set 6. It was apparent that the acetate-bound form was also present throughout data set 1 to data set 6 in addition to the conventional OH−-bound resting form of T2Cu. In this communication, we determined the acetate-free CueO structure (PDB entry 4ef3) and compared it with the previously published coordinates (PDB entry 4e9s; Kataoka et al., 2007 ▸), for which the presence of the acetate ion had not been discussed. Acetate ions easily reach T2Cu through the water channel, as demonstrated by the CueO crystals formed in the presence and absence of acetate ions.

2. Materials and methods  

The purification and crystallization of CueO were performed according to previously reported methods (Kataoka et al., 2007 ▸). The oxidizing activity of 2,2′-azinobis(3-ethylbenzo­thiazoline-6-sulfonic acid) (ABTS) was measured as reported in Kataoka et al. (2007 ▸) except for increasing additions of ammonium acetate. Citrate buffer was used for crystallization instead of acetate buffer (Table 1 ▸).

Table 1. Crystallization conditions for the acetate-free CueO.

Method Sitting-drop vapour diffusion
Plate type 24-well sitting-drop plate (Cryschem plate, Hampton Research)
Temperature (K) 293
Protein concentration (mg ml−1) 12
Buffer composition of protein solution 100 mM phosphate buffer pH 6.0
Composition of reservoir solution 100 mM sodium citrate buffer pH 5.6, 22% PEG 4000, 5% 2-propanol
Volume and ratio of drop (µl) 2:2
Volume of reservoir (µl) 500
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For data collection, CueO crystals were soaked in a cryoprotectant solution (10% glycerol) for a few minutes prior to cooling under a cold stream of nitrogen. X-ray diffraction data sets were collected at 100 K on beamline BL26B2 (λ = 0.8000 Å at SPring-8 using a MAR Research MX225 CCD detector (Ueno et al., 2006 ▸).

The unit-cell parameters were determined and reflections were integrated using HKL-2000 (Otwinowski & Minor, 1997 ▸) and the CCP4 program package (Winn et al., 2011 ▸). Using the reported structure of CueO (PDB entry 4ner; Komori et al., 2014 ▸), the molecular-replacement method was carried out. Further model building and structure refinement were performed using Coot (Emsley et al., 2010 ▸), REFMAC (Murshudov et al., 2011 ▸) and SHELX (Sheldrick, 2008 ▸). The progress and validity of the refinement process were checked by monitoring the R free value for 5% of the total reflections (Brünger, 1992 ▸). The data-collection and refinement statistics are summarized in Table 2 ▸. Model geometry was analysed using MolProbity (Chen et al., 2010 ▸) and no residues were found in the disallowed region of the Ramachandran plot. The figures were prepared using PyMOL (http://www.pymol.org). The coordinates have been deposited in the PDB with accession code 4ef3.

Table 2. Data collection and processing for the acetate-free CueO crystal.

Values in parentheses are for the outer shell.

Diffraction source BL26B2, SPring-8
Wavelength (Å) 0.8000
Temperature (K) 100
Detector MAR Research MX225
Crystal-to-detector distance (mm) 200.0
Rotation range per image (°) 1
Total rotation range (°) 180
Exposure time per image (s) 1
Attenuator Al, 1000 µm
Space group P21
a, b, c (Å) 50.28, 93.24, 59.59
α, β, γ (°) 90.00, 112.87, 90.00
Resolution range (Å) 17.60–1.90 (1.97–19.0)
No. of unique reflections 38421 (3579)
Completeness (%) 98.7 (92.1)
Multiplicity 3.7 (2.9)
〈I/σ(I)〉 10.4 (2.0)
R r.i.m. † 0.094 (0.343)
R 0.154 (0.213)
R free 0.184 (0.254)
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†

R r.i.m. was estimated by multiplying the conventional R merge value by the factor [N/(N − 1)]1/2, where N is the data multiplicity.

Tunnels leading from the protein surface to the TNC were visualized using Caver Analyst (Kozlikova et al., 2014 ▸).

3. Results and discussion  

In the previous structural analyses of CueO crystallized in acetate buffer we have shown changes in the structure around the TNC using six diffraction data sets collected under increasing X-ray dose conditions (PDB entries 4ner, 4e9q, 4e9r, 4e9s and 4e9t for data sets 1, 2, 4, 5 and 6, respectively; data set 3 was not deposited; Komori et al., 2014 ▸). The TNC for data set 1 obtained under low X-ray dose conditions had the O-centred structure with OH−-bridged T3Cu atoms and an OH−-bound T2Cu (∼50%). However, it was apparent that the acetate-bound T2Cu form was also present in place of OH− (∼50%; the presence of acetate ion was not mentioned in the previous crystal structure analyses). Fig. 2 ▸(a) shows the 2F o − F c map around T2Cu contoured at 1.0σ for acetate data set 1 (PDB entry 4ner) and Fig. 2 ▸(b) shows the F o − F c map around T2Cu contoured at 4σ for the same data set. Residual density is observable around the OH−-bound T2Cu, indicating the coordination of an acetate ion in the place of OH−. Fig. 3 ▸(a) shows the whole active site with the superimposition of forms representing both the binding of OH− and acetate ion. The distances between the cupric T2Cu and the two O atoms derived from the acetate ion are 2.2 and 3.1 Å, indicating monodentate coordination by the deprotonated hydroxyl group. The former bond distance is practically the same as that for the inorganic OH−-bound form, although the distances of the Cu2+—O− bond (for OH− and CH3CO2 −) were reported to be considerably shorter at <2 Å in small-molecule studies (Bazhanova et al., 2008 ▸).

Figure 2.

Figure 2

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(a) 2F o − F c map contoured at 1.0σ for acetate data set 1 (PDB entry 4ner). (b) F o − F c map contoured at 4.0σ for acetate data set 1 under low X-ray dose conditions (PDB entry 4ner). (c) 2F o − F c map contoured at 1.0σ for acetate data set 5 under high X-ray dose conditions (PDB entry 4e9s). (d) OMIT map of acetate contoured at 4.0σ for acetate data set 5 (PDB entry 4e9s). T2Cu and O atoms are shown as green and red spheres, respectively.

Figure 3.

Figure 3

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The active site of CueO crystallized in acetate buffer with OH− and acetate-bound T2Cu. (a) Acetate data set 1 under low X-ray dose conditions (PDB entry 4ner). (b) Acetate data set 5 under high X-ray dose conditions (PDB data 4e9t). Both the OH− and the acetate ion are shown together in (a) and (b). The central O atom is present in the centre of the TNC in (a), but has disappeared in (b), in which T2Cu and T3Cua are reduced by hydrated electrons. The large blue, green and orange spheres are the T1Cu, T2Cu and T3Cu atoms, respectively. The small red spheres are O2−, OH− or H2O.

The two other O species bound to Cu2+ are the central O2− inside the copper triangle and the OH− ion bridged between the T3Cu atoms (Fig. 3 ▸ a), both of which originate from the O2 co-substrate. Therefore, data set 1 collected under low X-ray dose conditions mainly contains a fully oxidized form of copper with O2− at the centre of the TNC: the intermediate II form or the native intermediate (the in situ-measured K-edge spectrum indicated that all Cu centres were cupric). With regard to the coordination geometry of T2Cu, the square-planar structure with the 2N2O ligand atom set (two N atoms from two His residues and two O atoms from the central O2− and OH− or acetate) is typical of cupric ion and differs from the T-shaped structure without the central O2− found in the classical resting form of MCOs.

Stepwise reductions of T1Cu, T2Cu and one of the T3Cu atoms (T3Cua) took place during the transformation from data set 1 to data set 6. Fig. 3 ▸(b) indicates the structural change induced at the TNC. The stepwise reduction of T2Cu and T3Cua were accompanied by protonation of the central O2− and the OH− bound to T2Cu and the subsequent elimination of an H2O molecule from the TNC through the water channel involving Asp112. Concomitantly, the d–π interaction between T3Cua and Trp139 became stronger owing to a decrease in the distance between T3Cua and the edge of the indole ring.

The acetate-bound form of T2Cu was present throughout all of the data sets. Figs. 2 ▸(c) and 2 ▸(d) show the 2F o − F c map contoured at 1.0σ for acetate in data set 5 (PDB entry 4e9s) and the OMIT map of acetate contoured at 4.0σ, respectively. The distances between the reduced T2Cu and the two O atoms from the acetate moiety were 2.5 and 3.6 Å. According to a small-molecule study, the bond distance between Cu+ ion and the O atom of acetate ion is ∼1.9 Å regardless of whether the structure is dimeric or tetrameric (Bazhanova et al., 2008 ▸). The binding of a protonated acetic acid molecule by Cu+ or Cu2+ has not been reported in small-molecule studies. A cage effect provided by a protein molecule might allow the presence of a protonated acetic acid near a cuprous ion, for which the geometry is linear (2N coordination) or T-shaped (2N1O coordination).

We have always utilized 100 mM ammonium acetate pH 5.2 with 20% polyethylene glycol (PEG 4000) and 5% 2-propanol to crystallize CueO and its mutants, which led to the formation of the acetate-bound form. To obtain unequivocal evidence that the acetate ion was derived from the buffer solution used for crystallization, we utilized a novel crystallization condition using citrate buffer at pH 5.6. As expected, the acetate-bound form was not observed even as a minor content in the crystals formed in citrate buffer (Fig. 4 ▸; PDB entry 4ef3; crystallo­graphic data are given in Tables 1 ▸ and 2 ▸). The coordinates of all atoms in Fig. 5 ▸ were practically the same as those in Fig. 3 ▸(a) except that acetate ion was never observed. The presence of acetate ion was not mentioned in the previous crystal structure analyses.

Figure 4.

Figure 4

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2F o − F c map contoured at 1.0σ for the citrate data (PDB entry 4ef3). T2Cu and O atoms are shown as green and red spheres, respectively.

Figure 5.

Figure 5

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The structure of the copper centre in CueO crystallized in citrate buffer without an acetate ion bound to T2Cu (PDB entry 4ef3). The crystals for X-­ray diffraction were obtained in citrate buffer pH 5.6, in contrast to those used for Fig. 1 ▸, which were obtained in acetate buffer pH 5.2. The central O atom is present in the centre of the TNC. The large blue, green and orange spheres are the T1Cu, T2Cu and T3Cu atoms, respectively. The small red spheres are O2−, OH− or H2O.

In the early studies on MCOs such as plant laccase, ascorbate oxidase and ceruloplasmin, exogenous small anions such as azide and fluoride were utilized to study the properties of the TNC (Koudelka & Ettinger, 1988 ▸). There is no doubt that these anions reach the TNC, in particular T2Cu, easily. It has also been supposed that the H2O molecule bound to T2Cu is deprotonated depending on the pH. T2Cu has frequently been observed to be depleted in many MCOs, suggesting that this copper centre is most easily accessible to solvent water among the four copper centres. Fig. 1 ▸ shows the presence of three channels leading from the protein surface to the TNC. The purple tunnel starting at the OH− coordinated to T2Cu is located between domain 1 and domain 3. Asp112 positioned to the side of it is hydrogen-bonded to the OH− ion coordinated to T2Cu via a water molecule and is also hydrogen-bonded to His101 and His448 coordinated to T2Cu and T3Cub, respectively. Mutation of Asp112 led to a reduction in enzymatic activity, indicating that this amino acid is one of the key amino acids in controlling the processes of O2 reduction and H2O excretion (Ueki et al., 2006 ▸). The purple tunnel has been considered to play a role in the excretion of H2O formed by the reduction of O2. Although we have not performed a soaking experiment with acetate, we conclude that the acetate ion in Fig. 2 ▸ has come from the buffer solution. The hydrogen-bond wire formed by water molecules and Asp112 is considerably dynamic and allows exogenous acetate ion, which is bulkier than linear anions such as N3 − and O2 2−, to reach the TNC and replace OH− or H2O coordinated to T2Cu (see above).

Exogenous acetate ions reach the TNC through the purple tunnel in Fig. 1 ▸ and presumably also approach the entrance of the tunnel to transport H+ (greenish-blue tunnel). Since Asp112 and Glu506 with a carboxy group in their side chains are involved in the former and latter tunnels, respectively, we determined the enzymatic activity of CeuO in phosphate buffer in the presence of 0–500 mM ammonium acetate. The oxidizing activity of CueO for ABTS was enhanced by the addition of up to 300 mM acetate ion, although it decreased at higher concentrations of acetate ion, presumably owing to the solution becoming slightly turbid (Fig. 6 ▸). In contrast to azide and halide ions, acetate ion is labile to substitution and does not inhibit the binding of O2 at the TNC. Furthermore, acetate ion might assist the transport of proton to TNC by existing in and near the channels leading from solvent to TNC. Further studies are required to ascertain whether this hypothetical supporting effect of proton transport by acetate ion prevails more widely over other MCOs.

Figure 6.

Figure 6

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Effect of the addition of ammonium acetate on the oxidizing activity for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) of CueO in 0.1 M phosphate buffer pH 6 at 25°C as determined from absorption changes at 420 nm. One unit of activity is the amount of enzyme required to oxidize 1 µmol substrate per minute. The pH values of the reaction mixtures were not changed on the addition of ammonium acetate. Three data points were averaged for each specific activity.

4. Conclusions  

The present results indicate that the channel formed between domains 1 and 3 in the CueO molecule is sufficiently wide and dynamic to allow exogenous acetate ions to approach T2Cu deeply buried inside the CueO molecule. An effect of addition of acetate ion in enhancing the enzymatic activity of CueO has also been observed.

Supplementary Material

PDB reference: CueO (citrate buffer), 4ef3

Acknowledgments

The present study was supported by Grants-in-Aid from Gakushin, Japan to TS (Nos. 26288076 and 2665031), a grant from the Inamori Foundation to HK and the fund for Kagawa University Young Scientists 2013 and 2015 (HK). We also thank the beamline staff at BL26B2 for their kind help with X-ray data collection.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

PDB reference: CueO (citrate buffer), 4ef3

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