Flexibility of the metal-binding regionin apo-cupredoxinsMaría-Eugenia Zaballaa, Luciano A. Abriataa, Antonio Donaireb,1, and Alejandro J. Vilaa,1

aInstituto de Biología Molecular y Celular de Rosario, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531,S2002LRK Rosario, Argentina; and bDepartamento de Química Inorgánica, Facultad de Química, Universidad de Murcia, Campus Universitario deEspinardo, Apdo. 4021, 30100 Murcia, Spain

Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved April 18, 2012 (received for review November 30, 2011)

Protein-mediated electron transfer is an essential event in manybiochemical processes. Efficient electron transfer requires the reor-ganization energy of the redox event to be minimized, which isensured by the presence of rigid donor and acceptor sites. Electrontransfer copper sites are present in the ubiquitous cupredoxin fold,able to bind one or two copper ions. The low reorganization en-ergy in these metal centers has been accounted for by assumingthat the protein scaffold creates an entatic/rack-induced state,which gives rise to a rigid environment by means of a preformedmetal chelating site. However, this notion is incompatible with theneed for an exposed metal-binding site and protein–protein inter-actions enabling metallochaperone-mediated assembly of the cop-per site. Herewe report an NMR study that reveals a high degree ofstructural heterogeneity in the metal-binding region of the nonme-tallated CuA-binding cupredoxin domain, arising from microsecondto second dynamics that are quenched upon metal binding. Wealso report similar dynamic features in apo-azurin, a paradigmaticblue copper protein, suggesting a general behavior. These findingsreveal that the entatic/rack-induced state, governing the featuresof themetal center in the copper-loaded protein, does not require apreformed metal-binding site. Instead, metal binding is a majorcontributor to the rigidity of electron transfer copper centers.These results reconcile the seemingly contradictory requirementsof a rigid, occluded center for electron transfer, and an accessible,dynamic site required for in vivo copper uptake.

metalloproteins ∣ nuclear magnetic resonance ∣ protein dynamics

Long-range electron transfer (ET) in proteins is a key chemicalevent in many essential biological processes such as cellular

respiration and photosynthesis (1–3). According to Marcus’ semi-classical theory, the outstanding efficiency of these processes isbased on the maximization of the superexchange coupling be-tween donor and acceptor and the minimization of the reorgani-zation energy, given the low driving forces found in mostbiological systems (4–6). Thus, with driving forces as low as 0.1 eVand distances larger than 10 Å, efficient long-range electrontransfer is only possible if the nuclear reorganization energy ofthe reactants is below 1 eV (4, 7).

Transition metal ions such as copper and iron are ubiquitous inETchains because their redox potentials and electronic structurescan be tuned by the protein environment to match the require-ments of different biological redox events. Cycling between thecoordination geometries preferred by these metals in each redoxstate would in principle lead to high reorganization energies (7).Electron transfer iron centers avoid this by using rigid cofactorssuch as iron-sulfur clusters and heme groups (8–10). Instead,copper ions in ETcenters are only bound to protein residues, asobserved in type 1 (blue) copper and CuA centers; and thus theprotein fold around the metal ion is expected to impart rigidity tothese otherwise flexible metal centers (10–12). Particularly, out-er-sphere coordination involving hydrogen bonding networks hasbeen proposed as responsible for the low reorganization energiesobserved in copper centers in proteins (13, 14). The strain intro-duced by the protein fold is then predicted to be responsible for

the unusual functional and spectroscopic features observed intype 1 copper and CuA centers (15). This scenario, known as theentatic/rack-induced state hypothesis, was originally formulatedfor protein-bound cofactors by Lumry and Eyring in 1954 (16)and then extended to electron transfer metalloproteins byMalmström and Williams in the late 1960s (17, 18). This concepthas been matter of debate along many years (7, 15, 19–26). Inparticular, Ryde et al. have reported in vacuum quantum calcula-tions suggesting the absence of any strain in the geometryadopted by the copper in the protein framework (21, 22).

The entatic/rack-induced state hypothesis applied to ETcopper proteins was strongly supported by several X-ray struc-tures of type 1 copper proteins revealing that the conformationof the copper ligands in the metal-depleted (apo) form was iden-tical to that found in the holoproteins (27–31). These resultsprompted the idea that the protein fold determines the coordina-tion geometry of the metal center by creating a preformedchelating site with very little flexibility, thus minimizing confor-mational changes that would normally be exhibited during Cu(II)/Cu(I) redox cycling (19). This idea is reinforced by the factthat all ETcopper centers are bound to a rigid Greek-key β-barreldomain, known as the cupredoxin fold (31–34). Instead, iron-sulfur and heme centers are found in different folding motifs,with diverse intrinsic mobilities (35–37).

The notion of a preformed rigid binding site is conflicting withthe finding that copper centers are loaded by specific metallocha-perones (38, 39). The concept of metallochaperones is relativelynew and it has replaced the idea that metal uptake dependsentirely on the chelating properties of the apoprotein. Severalcopper chaperones have been identified and characterized so far,requiring specific protein–protein interactions that can be metal-mediated in some cases (40). In the latter situation, the metal ionis simultaneously bound to ligands from both the metallochaper-one and the target protein during the transfer step (40). In anycase, the assumption of a highly rigid metal site, which should alsobe hidden from the bulk solvent to prevent unwanted side reac-tions, does not favor metal delivery by another protein. Thus, thedemands for efficient electron transfer and in vivo metallation arehard to reconcile given the current knowledge in the field.

We have recently elucidated the chaperone-mediated mechan-ism of copper uptake by the CuA-binding cupredoxin domain inThermus thermophilus ba3 cytochrome c oxidase (41). In thatwork, we observed that the NMR spectra of the apo and metal-

Author contributions: M.-E.Z., L.A.A., A.D., and A.J.V. designed research; M.-E.Z., L.A.A.,and A.D. performed research; M.-E.Z., L.A.A., A.D., and A.J.V. analyzed data; and M.-E.Z.,L.A.A., A.D., and A.J.V. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID code 2LLN). The NMR chemical shifts have been deposited in theBioMagResBank, www.bmrb.wisc.edu (accession nos. 18081 and 18254).1To whom correspondence may be addressed. E-mail: adonaire@um.es or vila@ibr.gov.ar.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1119460109/-/DCSupplemental.

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lated forms of this protein show significant differences, suggestingthat there might be nonnegligible structural perturbations uponmetal binding, consistent with the finding that copper uptake ismediated by a metallochaperone (Fig. S1). Here we report a de-tailed NMR study showing the solution structure and dynamicfeatures of this cupredoxin domain in the nonmetallated form.We have found that not only is the metal-binding site disorderedin the apoprotein but it also exhibits significant dynamics in themicrosecond to second timescale. Moreover, to prove that ourresults can be extended to other cupredoxins, we report an NMR-based study of Pseudomonas aeruginosa apo-azurin, which hasbeen considered as the paradigm of blue copper proteins andlong-range ET (42–44). Also in this case, we show there is signif-icant dynamics in the metal-binding region only in the absence ofthe copper ion. Thus, our findings indicate that the rigid coppercenter needed for efficient long-range ET is not preformed in thenonmetallated cupredoxin. Instead, metal binding does signifi-cantly contribute to the rigidity of these centers and the metal-binding region in the apoprotein is flexible enough to allow invivo metallation.

ResultsThe CuA-Binding Cupredoxin Domain. Subunit II of T. thermophilusba3 oxidase consists of a soluble CuA-binding domain anchored tothe membrane through an N-terminal transmembrane helix (32,45). This domain adopts a cupredoxin fold similar to the onefound in type 1 copper proteins, with a longer loop that allowsbinding of an additional metal ion (12). The two copper ions ofthe CuA site are bridged by two cysteine ligands (Cys149 andCys153), giving rise to a rigid Cu2S2 core. Each copper ion is alsocoordinated to a terminal histidine residue (His114 and His157)and a weak axial ligand: a methionine sulfur (Met160) and a back-bone carbonyl (Gln151), respectively. The soluble fragment here-in studied comprises only the soluble cupredoxin domain ofsubunit II, where the transmembrane helix is not part of the pur-ified protein. This fragment retains the structure observed in thewhole oxidase (32, 45) and it is competent for ETwith its biolo-gical redox partner (46, 47). Given that residues 1–42 are absentin our protein construct, residue numbering starts at 43 in theresults presented here, following that employed in the X-raystructure of oxidized holoCuA [Protein Data Bank (PDB) ID2CUA] (32).*

Resonance Assignment and Solution Structure of apoCuA.1H, 13C,

and 15N resonances were assigned for most residues in the non-metallated form of the soluble domain of T. thermophilus ba3oxidase subunit II (apoCuA, hereafter) (Table S1). Resonancescorresponding to eight non-proline residues were missing inthe 1H, 15N-heteronuclear single quantum coherence (HSQC)spectrum of apoCuA, whereas 19 residues presented duplicatedNH cross-peaks (see below). Five of the residues with missingresonances (Cys149-Gln151, His157, and Gln158) are locatedat the loop bearing five out of the six copper ligands (ligand loop,hereafter) whereas the remaining correspond to His114 (theN-terminal histidine coordinated to one of the copper ions), itsadjacent residue Gly115, and the N-terminal Met43. These eightresidues are thus involved in an exchange process whosefrequency lies right on an intermediate timescale that broadenssignals beyond detection. Among residues with duplicated reso-nances in the 1H, 15N-HSQC spectrum, five are located in theN-terminal region (Val44, Ile45, Ala47, Lys49, and Leu50) andduplication here arises from the cis-trans isomerization of Pro46in a slow regime, as reported for the reduced holoprotein [Bio-MagResBank (BMRB) entry 5819; ref. 48]. Most of the remain-

ing residues with duplicated correlations are located around themetal-binding site (Ala85, Ala87, Tyr90, Val112, Ile113, His117,Gly120, Asn124, Gly154, Gly156, Met160-Thr163). Comparisonof the assignments obtained for apoCuA with those availablefor the reduced holoprotein revealed that most chemical shiftperturbations also map close to the metal-binding site (Fig. 1and Fig. S1).

The calculated three-dimensional structure of apoCuA is welldefined by 2,147 (including 879 long-range) NOE-based distancerestraints and 215 experimental torsion angle restraints (Fig. S2and Table S2). The atomic rmsd values for the 20 models inthe refined structure are 1.0� 0.2 and 1.4� 0.1 Å for the back-bone and all heavy atoms, respectively. Considering only residuesfrom the rigid β-barrel domain (residues 53–148 and 162–168),the rmsd values for backbone and all heavy atoms are 0.49� 0.07and 1.01� 0.08 Å, respectively.

The apoprotein in solution adopts a β-barrel fold where mostregions are well-defined except the N terminus and loops 86–90and 149–161 (ligand loop) (Fig. 1 and Fig. S3). The disorderobserved at the ligand loop might be due to the lack of experi-mental constraints (Fig. S2) and/or true mobility of this region inthe absence of the metal ions. Indeed, the low number of con-straints in this region parallels the missing correlations in the1H, 15N-HSQC, suggestive of some degree of flexibility (seebelow). It is then clear that the polypeptide presents a similarGreek-key β-barrel fold both in the apo- and copper-loadedforms, and that the structural perturbations between both formsof the protein are confined to the metal-binding region, with theligand loop showing the highest degree of disorder.

Dynamics of apoCuA and Reduced holoCuA. Subnanosecond timescale.The dynamics of apo- and reduced holoCuA in the pico-to-nano-second timescale was studied by measuring 15N R1 and R2 and1H-15N heteronuclear NOE. Average correlation times of 8.3�0.1 ns (apoCuA) and 8.2� 0.1 ns (holoCuA) were estimated andused for relaxation data analysis with the model-free approach(49–51). Order parameter values (S2) for apo- and holoCuAare remarkably high along most of the sequence, averaging0.9� 0.1 in both proteins and confirming the high level of rigidityof the β-barrel (Fig. S4). As expected, the order parameter valuesfor both protein forms drop below 0.6 at the N terminus, due tothe high mobility usually observed in this region. S2 values lowerthan 0.6 are also observed for residues 154–156 in apoCuA. How-ever, a slight decrease in S2 values is also found in this region for

Fig. 1. NMR-based solution structure of T. thermophilus apoCuA. (A) Car-toon representation of the X-ray structure of oxidized holoCuA (from PDBID 2CUA; ref. 32). (B) Ribbon representation of the best 20 out of 80 structurescalculated by CYANA for apoCuA in solution (this work, PDB ID 2LLN). Regionswith significant chemical shift perturbations (Eq. S1) between apo- andholoCuA and structural disorder in the calculated structure of the apoproteinare highlighted in blue (ligand loop, residues 149–161), and orange (residues86–90).

*The PDB coordinates (PDB ID 2LLN) and resonance assignments (BioMagResBank entry18081) for apoCuA obtained during this work are deposited using the linear 1–126 num-bering.

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holoCuA. Overall, these data do not reveal significantly differentdynamic features in the pico-to-nanosecond timescale betweenthe apo and holo forms of the CuA-binding domain.

Microsecond to millisecond timescale. Protein motions in the micro-to-millisecond timescale were probed by constant-time Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion experi-ments (52, 53). Fig. 2A shows the ΔR2 profiles obtained forapoCuA and reduced holoCuA, where ΔR2 is the differencebetween R2 values measured at CPMG frequencies of 33 and966 Hz, validated by fitting of the dispersion curves to Eq. S2(Fig. 2, Inset and Fig. S5). The ΔR2 profile for apoCuA showstwo regions experiencing significant exchange in comparison tothe holoprotein. Both regions map close to the metal-binding site,as shown in Fig. 3A. Thus, residues in the metal-binding regionexhibit significant micro-to-millisecond dynamics in the apopro-tein, and this flexibility is lost upon binding of the metal ions.

Protein compactness.Protein compactness was studied by a 1H, 15Nheterogeneity-band-selective optimized-flip-angle short-transient-heteronuclear multiple quantum coherence (HET-SOFAST-HMQC) experiment with irradiation of aliphatic protons. TheλNOE parameter calculated from this experiment reports on theproton density around the probed amide, with low λNOE values(<0.3) indicating well-structured compact segments and highervalues indicating a less compact environment (which might bedue in turn to enhanced dynamics) (54). Fig. 2B shows the λNOEprofiles obtained for apo- and reduced holoCuA. Both proteinforms present a similar global compactness along most of the se-quence, except for a significant difference observed at the ligandloop, where the higher λNOE values obtained for the apoproteinsuggest a less compact structure in the absence of the copper ions.

Millisecond to second timescale.The absence (for eight non-prolineresidues) and duplication (for 19 residues) of NH cross-peaks inthe 1H, 15N-HSQC spectrum of apoCuA reveals chemical ex-change in the intermediate and slow regimes on the chemical shifttimescale, respectively. As detailed above, most of these residues

correspond to copper ligands and/or map to the surroundingsof the metal-binding site. We note that only one correlation isobserved for all these residues in the 1H, 15N-HSQC spectrumof reduced holoCuA (BMRB entry 5819). Thus, slow dynamicfeatures on the copper ligands and surrounding residues are onlyobserved in the absence of the copper ions (Fig. 3B).

Dynamics of apo-azurin. In order to extend these results to othercupredoxins, we have studied the solution dynamics of P. aerugi-nosa apo-azurin. The blue copper protein azurin has been exten-sively studied as a paradigmatic protein for electron transfer(42–44), and Cu(I)-azurin has been the subject of several NMRstudies (55–58). Instead, apo-azurin has not been characterizedby NMR.

Backbone 1H and 15N resonances were assigned for mostresidues of P. aeruginosa apo-azurin. Resonances correspondingto 17 non-proline residues were missing in the 1H, 15N-HSQCspectrum, indicating they are involved in an intermediate ex-change process on the chemical shift timescale. These residuesare Ala1-Cys3, Gly9-Asp11, Asn18, His35-Leu39, Gly45, His46,Gly88-Glu91, most of them mapping to the surroundings of themetal-binding site (Fig. 4B). This behavior contrasts with that ofCu(I)-azurin, whose 1H, 15N-HSQC spectrum shows one cross-peak for every non-proline residue in the protein (except theN-terminal Ala1) (56), indicating that this slow dynamic processtakes place only in the nonmetallated form, in agreement withthe findings on apoCuA. Moreover, most chemical shift perturba-tions resulting from metal removal in azurin cluster close to thecopper center (Fig. 4A and Fig. S6), again resembling the situa-tion met in the CuA-binding domain.

Protein dynamics in the micro-to-millisecond timescale wereevaluated by relaxation dispersion experiments in apo-azurin.As observed in Fig. 4C, the ΔR2 profile, validated by fitting ofthe dispersion curves to Eq. S2 (Fig. S7), shows three definedregions in the micro-to-millisecond timescale. Residues in theseregions are next to residues with missing correlations in the 1H,15N-HSQC of apo-azurin, thus evidencing a common exchangeprocess. Comparison of these data with those reported by

Fig. 2. Protein dynamics and compactness of apoCuA (black squares) and reduced holoCuA (gray triangles). (A) ΔR2 profiles showing dynamics in themicro-to-millisecond timescale. Fits of experimental data to Eq. S2 for some apoCuA residues are shown as examples in the inset of the figure (Gln91, circles;Met160, triangles; and Gly162, squares, showing significant relaxation dispersion; and Ala101, diamonds, located in a region showing no exchange). (B) λNOE

profiles showing proton density around the backbone amides.

9256 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1119460109 Zaballa et al.

Orekhov and coworkers for Cu(I)-azurin (55, 57) reveals an in-creased mobility at the metal-binding region in the absence of thecopper ion.

DiscussionIn this work, we present an NMR study on apoCuA, revealing thatthe metal-binding region (mainly the ligand loop) shows a disor-dered and less compact structure in the apoprotein compared tothe holo form (Figs. 1 and 2B) and that this disorder, in turn, isdue to a dynamic behavior of this region in the microsecond tosecond timescale (Figs. 2A and 3). The blue copper protein azurindisplays similar dynamic features in its apo form which also mapto the metal-binding region (Fig. 4). The absence of such dynamicfeatures both in holoCuA and Cu(I)-azurin allows us to concludethat this region is significantly flexible in the apoproteins and thatonly metal binding quenches the dynamics of these loops in thecupredoxin fold. Given the different loop length and structure inCuA and azurin, these results strongly suggest that this behavior isa general feature of cupredoxins.

Dynamics of Apo-Cupredoxins and the Entatic/Rack-Induced State.The entatic/rack-induced state in ET copper proteins refers tothe organized protein structure around the metal-binding sitethat ensures a low reorganization energy for efficient electrontransfer (7). There have been numerous reports and reviews sup-porting the entatic/rack-induced state hypothesis (7, 13, 19, 20,26, 59). On the other hand, theoretical and experimental studieshave questioned the role of the protein matrix in defining thegeometry of the copper center (21, 22, 24, 25). X-ray studies onthe apo form of several cupredoxins, showing no changes in theligands’ conformations upon removal of the copper ion, wereconsidered as experimental evidence of the entatic/rack-induced

Fig. 3. Mapping of the dynamic features in apoCuA. Residues experiencingdynamics in the absence of the copper ions (i.e., in apoCuA) are highlighted inthe structure of holoCuA (PDB ID 2CUA, ref. 32, used to evidence the proxi-mity of these residues to the metal ions). (A) Dynamics in the micro-to-milli-second timescale. Residues with ΔR2 < 1.3 s−1, ΔR2 > 1.3 s−1, and missingresidues are shown in blue, orange, and gray, respectively. (B) Slow dynamics.Residues with zero (missing), one or two (duplicated) correlations in the 1H,15N-HSQC spectrum of apoCuA are shown in red, gray, and yellow, respec-tively.

Fig. 4. NMR results on apo-azurin. Residues showing significant chemical shift perturbations or enhanced dynamic features are mapped on the tridimensionalstructure of Cu(II)-azurin (PDB ID 4AZU; ref. 33). (A) Chemical shift perturbations between apo-azurin and Cu(I)-azurin backbone amide resonances. Residueswith chemical shift perturbations ðCSPÞ < 0.2, 0.2 < CSP < 0.5, CSP > 0.5 are shown in blue, yellow, and red, respectively. Residues with missing NH correlationsare shown in gray. (B) Microsecond to second dynamics of apo-azurin. Residues with ΔR2 < 2 s−1, ΔR2 > 2 s−1 are shown in blue and orange, respectively.Residues with missing correlations in the 1H, 15N-HSQC spectrum are shown in red. In both representations, prolines are shown in gray. (C)ΔR2 profiles showingdynamics in themicro-to-millisecond timescale. Fits of experimental data to Eq. S2 for some apo-azurin residues are shown as examples in the inset of the figure(Ile87, circles; Met44, squares, showing significant relaxation dispersion; and Thr61, triangles, located in a region showing no exchange).

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hypothesis for ETcopper proteins (27–31). In addition, those re-sults put forward the idea that the protein matrix creates a pre-formed rigid chelating site in the absence of the metal ions, thusentirely determining the geometry of the copper center (19).

The present NMR-based results provide compelling evidenceof structural disorder, flexibility, and conformational exchange inthe metal-binding region of the apo form of the CuA-bindingcupredoxin domain. Flexibility and slow chemical exchange werealso observed here for apo-azurin, supporting the extension ofthese findings to other cupredoxins and contrasting the men-tioned X-ray studies. This discrepancy may be attributed to thefact that most of the reported structures of apo-cupredoxins wereobtained by crystallization of the holoprotein and subsequentmetal removal from the crystal by soaking with a chelating agent(28–30). Instead, direct crystallization of apo-azurin gave rise tosome (minor) structural heterogeneity selectively on the copperligands (27). However, analysis of the structure of apo-azurindoes not reveal a significant increase in the B factors in the metal-binding region nor the lack of electron density, suggesting that theabsence of dynamics in these cases is due to crystal packing.

We note that our results do not necessarily support nor refutethe entatic/rack-induced state hypothesis because they apply tothe nonmetallated protein and do not assess the strain the proteinenvironment exerts on the Cu(I) and Cu(II) centers. Instead, ourfindings show that the entatic/rack-induced state, when operativeon the metallated protein, does not require a preformed rigidchelating site. This conception is in line with the results reportedby Wittung-Stafshede and coworkers, showing that a rigid, ET-functional blue copper center can be obtained from Cu(II) bind-ing to unfolded azurin, suggesting that the constraints imposed bythe protein to the geometry of the metal center do not require apreorganized binding site defined in the polypeptide (60–62).

Dynamics of Apo-Cupredoxins and in Vivo Copper Uptake.The coppercargo in the CuA-binding cupredoxin domain of T. thermophilusoxidase is due to the action of a specific Cu(I)-metallochaperone(PCuAC) (41). Similar copper-loading mechanisms have beenproposed for the type 1 protein plastocyanin (63). Copper trans-fer mediated by chaperones generally takes milliseconds to sec-onds, overlapping with the fluxional timescales measured in thiswork for the metal-binding region of apoCuA and apo-azurin(although no copper chaperones have been reported for azurinto date). This observation suggests that the observed flexibilityof this region in the apo form of the protein allows the cupredoxinfold to sample different conformations, enabling copper transfer.

Our unique picture of a folded apo-cupredoxin consisting in aβ-barrel with disordered flexible loops is fully consistent with theneed of specific protein–protein interactions and defines an ex-

posed, dynamic metal-binding site allowing for efficient metallo-chaperone-mediated copper binding in vivo. In turn, the fullyconserved Greek-key β-barrel scaffold would be able to rendera rigid and efficient ET site once the copper ions are bound.Our results thus allow us to reconcile the seemingly contradictoryrequirements of a rigid, occluded center for electron transfer, andan accessible, dynamic site for in vivo copper uptake. Nature hassolved this problem by exploiting the cupredoxin fold, which isendowed at the same time with rigid and dynamic features thatare finely tuned by the binding of the metal ions.

Materials and MethodsProtein Preparation. Samples of azurin and apo- and holoCuA proteins wereuniformly labeled with 15N or 13C and 15N (Cambridge Isotope Laboratories,Inc.) and produced as described elsewhere (40, 47, 64). The apoCuA proteinwas expressed and purified in the absence of copper, whereas azurin waspurified as Cu(II)-azurin and the apoprotein was obtained by dialysis against50 mM KCN, 200 mM Tris·HCl pH 8.5, followed by several dialysis steps with-out the complexing agent. Protein samples for NMR experiments were pre-pared in 100 mM phosphate buffer pH 7.0 for azurin and pH 6.0 for the CuA

domain, adding 100 mM KCl in the case of the holoprotein or 2 mM DTT forthe apoproteins in order to avoid oxidation of the cysteine ligands. Proteinconcentration was about 1 mM.

Nuclear Magnetic Resonance Spectroscopy.NMR experiments were carried outon a 600 MHz Bruker Avance II Spectrometer equipped with a triple reso-nance inverse (TXI) probe head and on a 900 MHz Bruker Avance II Spectro-meter equipped with a triple resonance inverse (TCI) cryoprobe. Allexperiments were carried out at 298 K using standard techniques, as de-scribed in SI Text. The obtained family of structures for apoCuA is depositedat the Protein Data Bank under PDB ID 2LLN. Chemical shifts for all 1H, 13C,and 15N nuclei assigned in this work are deposited at the Biological MagneticResonance Data Bank under accession numbers 18081 and 18254 for apoCuA

and apo-azurin, respectively. Resonance assignments for the reduced metal-lated proteins, holoCuA and Cu(I)-azurin, were taken from the literature (48,56) and transferred by us to the sample conditions used in this work.

ACKNOWLEDGMENTS. The authors thank J. H. Richards (Caltech) for providingthe plasmid for azurin expression. The Centro di Risonanze Magnetiche ofFlorence, Italy, and the European Program Bio-NMR (BIO-NMR-00012) areacknowledged for the access to high magnetic field spectrometers. A.D.also thanks the Spanish Ministerio de Educación for a grant supporting afour months’ stay in Rosario, Argentina (PR2009-0479). M.E.Z. and L.A.A. aredoctoral and postdoctoral fellows from Consejo Nacional de InvestigacionesCientíficas y Técnicas (CONICET), respectively. A.J.V. is an Howard HughesMedical Institute International Research Scholar and a staff member fromCONICET. The Bruker Avance II 600 MHz was purchased with funds fromAgencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and CON-ICET. This research was funded by ANPCyT, Argentina (Proyecto de Investiga-ción Científica y Tecnológica 2007-314), the Spanish Ministerio de Economía yCompetitividad, and Fundación Séneca de la Región deMurcia, Spain (projectnumbers SAF2011-26611 and 15354/PI/10, respectively).

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