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J. Biol. Chem., Vol. 278, Issue 34, 31958-31963, August 22, 2003
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**
From the
Departments of Biochemistry and Molecular
Biophysics and ¶Soil, Water, and Environmental
Science, University of Arizona, Tucson, Arizona 85721
Received for publication, March 24, 2003 , and in revised form, May 30, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Both the cue and pco systems rely on multicopper oxidases
for copper tolerance; however, the functional role of these proteins remains
unclear, as does the in vivo substrate. In general, multicopper
oxidases couple four one-electron substrate oxidation steps to the
four-electron reduction of dioxygen to water
(14). CueO can oxidize a range
of substrates in vitro, including catechols, siderophores, and Fe(II)
(10), and this activity is
greatly enhanced in the presence of excess (
100 µM)
copper(II) ions (10,
15), suggesting that binding
of a labile copper is important for enzymatic activity. We recently determined
the crystal structure of CueO at 1.4-Å resolution
(15) and showed that its
structure is similar to that of other multicopper oxidases, such as ascorbate
oxidase (16) and several
laccases
(17–19).
As expected, CueO contains a T1 "blue copper" site, near the point
at which the larger substrate is believed to bind, as well as a trinuclear
copper center, where oxygen reduction occurs. However, CueO possesses a
methionine-rich motif not found in these other proteins that could potentially
be involved in copper binding. Portions of this motif (residues 356–371)
are part of a helix lying near, and blocking access to, the T1 copper site
(15); the remaining residues
in the motif (380–402) are disordered in the structure.
Methionine-rich regions are found in numerous proteins involved in copper homeostasis, leading to the suggestion that such regions are involved in copper binding (recently reviewed in Refs. 20 and 21). Of these, Ctr1 (for copper transport protein 1) has been shown to require one or more MXXM motifs for activity in the yeast and human proteins (22), and CopA has been shown to bind seven extra copper ions (23). However, PcoC and the related periplasmic protein CopC bind only one copper ion (20, 21, 23). The recently determined PcoC crystal structure displayed an interface between methionine-rich regions in adjacent molecules in the crystal, leading the authors to suggest that the role of such regions may be in protein-protein recognition, possibly between PcoC and PcoA, rather than in copper binding (24).
In the present work, we report the crystal structure of CueO bound to exogenous copper. We find a copper ion bound to the methionine-rich helix near the T1 center, ligated to residues required for copper tolerance. Mutations to the ligating residues yield reduced CueO oxidase activity in vitro and reduced copper tolerance in vivo.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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X-ray Data Measurement and Structural Refinement—For the CuCl2-soaked crystal, data were measured at Stanford Synchrotron Radiation Laboratory beam-line 9–2 and processed with D*TREK (25) (Table I). An anomalous difference Fourier electron density map calculated with phases from the CueO model with copper atoms deleted revealed the four expected copper sites, an additional highly occupied site near the T1 copper, and a partially occupied site on the surface of the protein. These atoms were added to the model and the structure refined with SHELX (26). Metal-ligand distances and angles were not restrained. Refinement cycles were interspersed with rebuilding using O (27). Final refinement included hydrogen atoms in calculated positions. Other programs of the CCP4 package (28) were used for structural analysis, and MolScript (29), Bobscript (30), and Raster3D (31) were used for figure preparation. Data for the M441L mutant were measured at the Advanced Photon Source, BioCARS, beam line 14-BMC. Data were processed with D*TREK and refined with REFMAC from the CCP4 suite (28).
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Bacterial Strains and Growth Media—Strains used in this work
are E. coli XLI blue, GR15 
cueO
cusCFBA (pASK-IBA3), GR15 (pCueO)
(9), GR15 (pCueO M355L), GR15
(pCueO M441L), GR15 (pCueO D360A), and GR15 (pCueO D439A). E. coli
cells were grown in Luria Bertani medium with antibiotics (100 µg/ml
ampicillin and 15–20 µg/ml chloramphenicol) or with CuCl2
added where appropriate.
Mutagenesis of cueO—Chromosomal DNA of E. coli strain W3110 was purified using Genomic-tips (Qiagen). Mutant cueO derivatives were generated by overlap extension-PCR from genomic DNA of E. coli W3110 with the Expand High Fidelity PCR system (Roche Applied Science) using the following primers: FW, 5'-GAAGAATTCATGCAACGTCGTGATTTCTTAAAAT-3' with reverse primers M355L-U, 5'-TCGAGCATCGGGTCCAGAGAGAGTTGCAGCTTG-3'; D360A-U, 5'-CATCTGCATCCCCATCATAGCGAGCATCGGGTC-3'; D439A-U, 5'-GGATGCAGCATCATGGCGCCCACGCCAGAGATA-3'; M441L-U, 5'-TATGGAACGGATGCAGCAGCATGTCGCCCACGC-3' yielding upper partial cueO genes and REV, 5'-GTCCTGCAGTACCGTAAACCCTAACATCATCCCC-3' with forward primers M355L-D, 5'-CAAGCTGCAACTCTCTCTGGACCCGATGCTCGA-3'; D360A-D, 5'-GACCCGATGCTCGCTATGATGGGGATGCAGATG-3'; D439A-D, TATCTCTGGCGTGGGCGCCATGATGCTGCATCC-3'; M441L-D, GCGTGGGCGACATGCTGCTGCATCCGTTCCATA-3' yielding lower partial cueO genes (restriction sites are underlined and base changes are bold). Cognate PCR fragments with introduced base pair changes were mixed, treated with DpnI to digest the dam-methylated template DNA, purified, and used for a second round of PCR with primers FW and REV. The mutated full-length cueO genes were purified, cut, and cloned into plasmid pASK-IBA3 (IBA, Göttingen, Germany) via the EcoRI and PstI restriction sites and transformed into E. coli XLI blue (Stratagene). The resulting plasmids expressed cueO-derivatives as C-terminal fusion proteins with the Strep-TagII epitope (SAWSHPNFEK), plus eight additional residues at the N terminus (GDRGPEF), which are removed along with the next 27 amino acids that comprise a signal sequence during export to the periplasm (15).
Immunoblotting—To detect CueO, strains expressing CueO-derivatives were grown in Luria Bertani broth at 37 °C to A600 = 0.5. The inducer anhydrotetracycline (200 µg/l) was added and CueO-expression was carried out for 3 h at 30° C. Cells were harvested, aliquots separated on SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane. CueO-derivatives were detected by their Strep-TagII-epitopes with a streptavidin-horseradish peroxidase conjugate, as described previously (13).
Functional Assays—The model substrate 2,6-dimethoxyphenol (DMP)1 (Aldrich) was used to assess catalytic activity by monitoring the change in absorption at 468 nm in the presence of CueO, as described previously (9). CueO activity was also measured as oxygen uptake in presence of external CuCl2 using DMP as an artificial electron donor. The change in oxygen concentration was monitored by an oxygraph instrument (Hansatech, Cambridge UK), as described previously (32). The assay mixture contained 0.1 M Tris buffer (pH adjusted with glacial acetic acid to specified values), 2 mM DMP, variable concentrations of CuCl2, and 250 µM O2 (estimated from Table values) at room temperature. Purified CueO protein was added to reach a concentration in the range of 25–100 µg/ml. The reaction was initiated by the addition of CuCl2 (iron content < 0.005%; Mallinckrodt AR) and the initial rates were determined. Kinetic values for Vmax and Km were estimated using Lineweaver-Burk analyses and varying copper concentration between 0.14 and 20 mM. Water was millipore-purified and solutions were freshly prepared before each experiment.
Mass Spectroscopy—Mass spectral analyses were performed on a
Thermoquest LCQ and a Bruker Reflex III MALDI-TOF instrument. Samples measured
by MALDI-TOF were mixed with a saturated solution of
3,5-dimethoxy-4-hydroxycinnamic acid in 70:30 H2O/acetonitrile
containing 0.1% of TFA, in such a way that the total amount of protein
deposited was 1–2 pmol. In general, a laser attenuation of 80% was used.
Electrospray ionization experiments were performed by direct injection of a
10 µM solution of the protein in MeOH/H2O, 1:1,
containing 2% AcOH. All protein samples analyzed were in 20 mM
Tris, pH 7.5, but diluted accordingly.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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= 1.378 Å to maximize anomalous
scattering by the copper atoms. The anomalous difference Fourier electron
density map, using phases calculated from a CueO model with copper atoms
removed, displayed six strong peaks, five of which are shown in
Fig. 1. Four of the strong
peaks (28–30
) coincide with the previously defined T1 and
trinuclear copper positions, whereas a fifth strong site (18) is
located near the T1 copper position and is consistent with a fully occupied
copper atom, referred to below as the labile or regulatory copper. A weaker
peak (11) is apparently the result of a partially occupied
copper-binding site on the surface of the protein that is unlikely to be of
functional importance.
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In the refined structure, the labile copper atom lies 7.5 Å from the T1 copper and is buried under the methionine-rich helix (Fig. 1). Coordination is through the SD atoms of Met-355 (2.2 Å) and Met-441 (2.5 Å), the OD1 atoms of Asp-360 (2.4 Å) and Asp-439 (2.1 Å), and a water molecule, HOH 2301 (3.1 Å), in a distorted trigonal bipyramidal coordination geometry (Fig. 2). Met-355 and HOH 2301 occupy axial positions, and Met-441, Asp-360, and Asp-439 are equatorial ligands. Interestingly, the labile copper and the T1 copper are directly linked through a hydrogen-bond from OD2 of Asp-439 to NE2 of His-443. As noted above, these two residues ligate the labile and T1 copper atoms, respectively, and the hydrogen-bond between them may provide a pathway for electron transfer between the two copper atoms. The largest structural change is for the side chain of Asp-439 with the ligating atom OD1 moving 2 Å, and CB moving 1.75 Å. The overall temperature factors for the labile copper and coordinating residues are somewhat higher than those for the other copper centers, averaging about 33 Å2 versus 22 Å2 for the entire protein (25 Å2 for side chain atoms), but the positions for these atoms are clearly defined in the final electron density maps. In addition, there is no residual electron density in the former position of the Asp-439 side chain.
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Protein Conformation—There are no significant structural
changes in the protein upon copper binding. The coordinating side chains
reorient to accommodate copper ligation, which is associated with small shifts
in nearby backbone positions. The overall root-mean-square deviation in
C
atom positions between the structures with and without added copper
is 0.18 Å; the largest shift (0.84 Å) was for the ligating residue
Asp-439. It is possible that crystal lattice contacts could mask larger
changes in this region upon binding copper, given that the structure was
obtained via soaking of existing crystals with CuCl2;
however, this seems unlikely because of the sparse number of contacts in the
region. Furthermore, the crystals have oxidase activity.
As noted, the labile copper ion is buried under the methionine-rich helix (residues 356–371), which, as in the absence of the labile copper, contains several non-copper-ligating methionine residues with poorly ordered side chains. The disorder is particularly evident in the methionine SD and CE atoms, where very large temperature factors are displayed. Amino acids 380–402 are not visible in the present electron density maps, as was also true in the absence of the labile copper. We have confirmed, by MALDI mass spectroscopic analysis of the frozen, copper-soaked crystals as prepared for data collection, that the protein is intact and not cleaved in this region. Possibly, the 22 unobserved amino acids become ordered in the presence of substrate or are involved in as-yet-unknown protein-protein interactions. A sixth partially occupied copper atom is found on the surface of the protein away from the active site region (15 Å from the T2 copper) and bound to His-488, Asp-132, and HOH 2001.
Trinuclear Copper Cluster—In the initial report of the
crystal structure of CueO, we noted an unusual trinuclear center geometry
containing a linear copper-oxygen-copper geometry
(15). The copper atoms of the
trinuclear center lie in nearly identical positions in the previous and
present structures; however, in the present structure, modeling of the
bridging atom as a single oxygen resulted in a low temperature factor (3
Å2) and a 5 residual electron density peak, indicating
the need for more electrons at this site. The density is clearly spherical,
and modeling as dioxygen, as was done in the structure of laccase from
Melanocarpus albomyces
(19), resulted in an
unsatisfactory refinement. Modeling the atom as chlorine (10 mM
CuCl2 was added to the crystal), refined well, yielding
copper-to-chlorine distances of 2.3 and 2.5 Å (unrestrained), similar
temperature factors for copper and chlorine atoms (20
Å2), and no residual electron density. The modeled geometry
is appropriate for a copper-chlorine-copper moiety. Furthermore, this chlorine
is 3 Å from the T2 copper and completes a square planar coordination
sphere for that copper ion.
We have reinvestigated the identity of the bridging atom in the original structure and both oxygen and chlorine refine satisfactorily in this position with acceptable temperature factors; the oxygen temperature factor was somewhat, but not dramatically, low (14 Å2), and the chlorine temperature factor was high (30 Å2) compared with the average of the copper atoms of the trinuclear cluster (14, 22, and 30 Å2). Because the geometry of the trinuclear cluster we observed is that expected for a bridging chloride ion, we now believe that the structure previously reported contains a bridging chlorine atom in the trinuclear cluster.
Mutagenesis and CueO Activity—To investigate whether the
labile copper atom contributes to CueO activity, four mutants were constructed
(M355L, D360A, D439A, and M441L), each of which has one ligand removed from
the copper coordination sphere. The UV-visible absorption spectra are the same
for the wild-type protein and the M355L, D360A, and D439A proteins, confirming
that these mutations have not affected copper incorporation into the nearby T1
copper site. However, for the M441L protein, the absorbance at 610 nm,
relative to that at 280 nm, shows about 50% less intensity, suggesting that
the mutation has affected copper incorporation into the T1 copper site. X-ray
diffraction data for the M441L mutant protein has been measured and the
structure analyzed. The M441L polypeptide is identical with that of the
wild-type protein (Protein Data Bank code 1KV7
[PDB]
) except for the replacement of
the side chain at the mutation site. However, a large negative peak
(–10) appears centered at the T1 copper site. Setting the
occupancy of the T1 copper to one half results in the disappearance of this
negative peak, indicating that the T1 copper is about half depleted, in
agreement with the spectroscopic results.
None of the mutant CueO proteins were as effective as wild-type CueO in protecting cells against copper-induced cell death; two of them, M355L and D360A, are completely inactive (Fig. 3). Oxidase activity of the purified mutant proteins is also decreased by the mutations (Table II). Under the conditions used (pH 6.8, 200 mM Tris-acetate buffer), all mutant CueO proteins show significantly lower oxidase activity; one, M355L, shows essentially no activity. Thus, disruption of the labile copper-binding site has led to a reduced copper oxidase specific activity in vitro and loss of the copper tolerance phenotype in vivo. D439A is the most active of the four mutant proteins, in agreement with the results of the copper tolerance experiments.
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To assess whether the mutations led to proteins with altered stability, we examined oxidase activity after incubating at elevated temperatures. Wild-type CueO and the four mutant proteins were subjected to 10-min incubations, ±2 mM CuCl2, at 30, 40, 50, 60 and 70 °C, followed by measurement of their activities at 25 °C. Wild-type CueO is reasonably thermostabile, displaying 67% of its activity after 70 °C incubation in the absence of added copper and 91% in the presence of excess copper. Thus, binding of the labile copper has a small stabilizing affect on the protein. Mutants M355L, M441L, and D439A retained 93, 60, and 63% of their original activity, respectively, after incubation at 70 °C in the presence of excess copper, and thus display stabilities similar to those of wild-type CueO but without the added stabilization through labile copper binding (the value for M355L is less precise because of the very low inherent activity in this protein). Mutant D360A was found to be more thermolabile and displayed only 32% activity after incubation. In the physiological temperature range of 25 to 37 °C, only small losses in activity were observed for any of the proteins; thus, we conclude that the altered kinetics of the mutants is predominately caused by loss of binding at the labile copper site.
The pH dependence of the CueO reaction with oxygen was measured for the wild-type enzyme and the four mutants. The initial reaction rates were determined in the pH range of 4.8–9.0 (Fig. 4). The wild-type enzyme has a broad activity peak between pH 5 and 6 and a second activity peak at pH 8 that was not observed for the mutants. The pH dependence of the reaction as monitored by oxygen consumption is different from that reported previously for the colorimetric determination of DMP oxidation. Oxygen consumption is directly linked to enzyme turnover and is therefore likely to provide a more reliable rate estimate than the indirect measurement of rates through a colorimetric assay based upon the polymerization of DMP oxidation products.
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In both in vitro and in vivo measures of CueO activity, the mutants located on the methionated helix, M355L and D360A, are the most impaired; M355L is almost inactive. The mutants of the copper-binding residues nearest the trinuclear center, D439A and M441L, retain considerably more activity, despite the M441L mutant apparently having a somewhat depleted T1 copper site.
Effect of Chloride on Oxidase Activity—To investigate whether the bridging chloride ion affected enzyme activity, oxygen consumption was measured in the presence and absence of chloride. CueO apo-protein was isolated under chloride-free conditions in Tris·SO4 buffer. Oxygen consumption was measured as described above in Tris·SO4 buffer, pH 8.0, using 2 mM DMP and 22.5 µg/ml copper-free apo-protein. The reaction was initiated by the addition of 1 mM CuSO4 or CuCl2. No lag phase was observed in oxygen consumption, indicating that reconstitution occurs quickly on the time scale of this experiment. A rate of 2.9 µM O2/mg protein/min was observed for the CuSO4 reconstituted protein, comparable with that seen for the enzyme reconstituted with chloride (3.3 µM O2/mg of protein/min).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Regulatory Copper Ligation Geometry—Copper ions in proteins have been found bound to the peptide chain via histidine, cysteine, methionine, carboxylic acid (Asp, Glu), and N-terminal amide ligation, but histidine ligation is by far the most common (33). Ligation sites that do not include histidine are rare; only three examples have previously been described (24, 34, 35). Thus, the finding that methionine and aspartate residues coordinate the regulatory copper is surprising. The regulatory copper is probably in the Cu(II) oxidation state, given that carboxylic acid ligands are considered "hard" and do not bind to Cu(I) in this fashion. Furthermore, trigonal bipyramidal complexes of the sort found for rCu are well known for Cu(II), whereas Cu(I) prefers lower coordination numbers. The buried rCu site is also unlike those for the copper chaperone proteins, where the ligation site is on the protein surface and copper is ligated by cysteine residues (36).
The present structure also displays an unexpected bridging chloride ion at the trinuclear center, apparently resulting from the high chloride concentration in the crystal. This chloride bridge apparently does not affect catalysis, because we measure similar activity for CueO in the presence or absence of chloride. Furthermore, preliminary structural data with chloride-free protein indicate that a bridging oxygen occurs with a trinuclear center geometry similar to that found in ascorbate oxidase (16).
Functional Role for rCu—The major difference between the structure of CueO and that of the related proteins ascorbate oxidase and laccase is the methionine-rich insert located near the T1 copper site. In ascorbate oxidase and laccase, the T1 copper lies near the surface of the protein, whereas in CueO, the T1 site is buried under a methionine rich helix that limits access to substrate. The binding of rCu at this site, only 7.5 Å away and linked to the T1 copper through a hydrogen-bond, suggests that rCu may have a catalytic role in the protein. Certainly, binding of the copper at the rCu site alters activity, allowing catalysis, as previously described, but also interfering with direct reduction of the T1 copper by photoactivated deazariboflavin (15).
Interestingly, labile M(II) binding sites have been observed in ceruloplasmin, a human multicopper oxidase found in plasma with ferroxidase activity (37). Ceruloplasmin, in its normal state, contains six Cu atoms: the four copper atoms usually seen in multicopper oxidases and two additional copper ions in T1-like sites. Two more labile M(II) binding sites have been identified, one of which lies in domain 6 and is analogous to the labile site we find in CueO. Mutagenesis of two of the ligands to the domain 6 ceruloplasmin labile M(II) binding site reduces ferroxidase activity by half (38), suggesting that Fe(II) binding at the labile sites is a step in iron oxidation.
Although CueO also shows ferroxidase activity in vitro, it is unlikely that the mechanism proposed for iron oxidation in ceruloplasmin is operative in CueO. CueO shows only slight ferroxidase activity in vitro unless Cu(II) is present in solution and, presumably, bound at the rCu binding site. Furthermore, the ligand set of the labile Cu seems not to be favorable for Fe(II) binding.
The true substrate for CueO remains unknown; however, biochemical (10) and genetic2 evidence is accumulating that suggests enterobactin, an iron siderophore, is the true substrate. Enterobactin oxidation possibly interferes with copper receptor mechanisms or leads to a copper-binding product.
Role of the Methionine-Rich Region—Two of the rCu-ligating residues, Met-355 and Asp-360, lie at the beginning of the methionine-rich helix-loop that covers the entrance to the T1 copper site. Mutation of these residues causes a bigger loss of CueO activity than mutation of the residues nearer the T1 copper. Insertion of the T1 copper into the protein seems not to be affected by these residues, unlike the M441L mutation. Aside from these residues involved in rCu ligation, none of the other nine methionines we see in the helix-loop region are altered in the present structure, although all of the side chains are disordered in both CueO structures. Furthermore, the remaining five methionines that lie in the disordered portion of the methionine rich region remain disordered. These results suggest that the methionine-rich region in CueO, and by extension in other proteins, is not present simply to bind copper. Other possible roles in CueO include involvement in substrate binding or in mediating protein-protein interactions, as has been suggested for PcoC (24).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL62969 and
GM58727 (to W. R. M.). Work performed at the Advanced Photon Source was
supported by the U. S. Dept. of Energy, Sector 14, BIOCARS, and by National
Institutes of Health Grant RR07707. The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked "advertisement" in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact. 
Permanent address: Lehrstuhl für Biochemie der Pflanzen,
Ruhr-Universität Bochum, D-44780 Bochum, Germany.
|| Present address: Institut für Mikrobiologie,
Martin-Luther-Universität Halle-Wittenberg Kurt-Mothes-Str. 3, 06099
Halle, Germany.
** To whom correspondence should be addressed: 1041 E. Lowell St., Tucson, AZ 85721; Tel.: 520-621-1884; Fax: 520-621-1697; E-mail montfort{at}email.arizona.edu.
1 The abbreviations used are: DMP, 2,6-dimethoxyphenol; MALDI-TOF,
matrix-assisted laser desorption ionization time-of-flight.
2 G. Grass and C. Rensing, unpublished observations.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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), pCueO M355L (
), pCueO D360A
(
), pCueO D439A (
), pCueO M441L (
), or pASK-IBA3 (•).
Shown are the averages of three independent experiments. Inset,
immunoblot against Strep-TagII-labeled CueO mutants from crude extracts of the
different cueO-derivatives, indicating a high level of expression for
the mutant proteins The lanes contain GR15 (
, M355L; 
, D360A;