The Copper Active Site of CBM33 Polysaccharide Oxygenases

Protein Isolation, IsothermalCalorimetry, and Thermal Stability

The Bacillusamyloliquefaciens CBM33domain, (first described as chitin-binding protein ChbB from a chitinolyticstrain of B. amyloliquefaciens;³⁰ hereafter BaCBM33), was producedfrom expression of the codon-optimized gene and subsequent periplasmicsecretion in E. coli allowing processingof the signal peptide to yield the free N-terminal histidine. Standardpurification with Q Sepharose and Superdex columns yielded good quantitiesof pure protein.

Copper(II) binding to apo-BaCBM33 was followed using isothermal titration calorimetry(ITC) at 298 K, at pHs 5, 6, and 7. Copper binding has an apparent K_D of 6 nM at pH 5 (c value>1000), which is comparable to the value of 55 nM recently foundforthe binding of Cu(II) to CBP21.²⁰ Copper(II)affinity is lower at higher pH giving K_D values of 40 nM at pH 6 (c value = 325) and 80nM at pH 7 (c = 435), with a copper to protein stoichiometryof approximately 0.8:1 (Figure 2). The smallincrease in K_D with pH is possibly dueto competition for copper binding from the buffers used at higherpH or potentially from the deprotonation of active site residues involvedin the stabilization of a metal-bound water molecule (see below forfurther discussion), although the reasons for these differences awaita more detailed binding study. In either case, binding of copper tothe enzyme is rapid and tight.

Figure 2

Isothermal calorimetry of metal bindingto BaCBM33:(A) Cu(II) at pH 6 (see Figure S1 for otherpH measurements), (B) Zn(II) at pH 5.

Binding of nickel(II), manganese(II), and zinc(II) at pH5 wasalso investigated using ITC, with Zn(II) the only metal showing anymeasurable binding (Figure 2). To corroborateITC data, the thermostability of the protein was measured in the presenceof these metals. In the presence of Cu(II), the melting temperature(T_m) increased by 20 K (Figure S2), while in the presence of Ni(II) and Zn(II), aless significant shift of 7 K was observed reflecting the lower affinityof CBM33 for these metals. Mn(II) had no impact on the T_m, suggesting no binding at all. The potential bindingof manganese ions was of interest because there is a single reportof GH61 using Mn as a metal ion in the absence of copper.¹⁶ However, in the case of BaCBM33,no Mn binding could be detected, strongly suggesting that CBM33s areuniquely copper enzymes. We also note that the enzyme does not bindfurther copper ions with any significant binding constant, analogousto GH61s and commensurate with the active site in CBM33s being a mononuclearcopper active site, and in contrast to the dicopper site proposedfor copper methane monoxygenase in which the histidine brace alsoappears. Such selective binding of a single copper corroborates therecent reports from activity assays that CBM33s should be classifiedas mononuclear copper-dependent oxygenases.¹⁹

Three-Dimensional Structure from X-ray Diffraction

To investigatethe nature of the structure of BaCBM33 in more detailand, in particular, the copper site, the three-dimensionalstructures of BaCBM33 in apo, copper-bound,and copper-bound/ascorbate-soaked forms were solved by molecular replacementat resolutions of 1.8, 1.9, and 1.7 Å, respectively. The structuresare analogous to those already reported for CBM33 and GH61 consistingof a core immunoglobin-like β-sandwich domain with an adjacenthelical bundle (defined as loop L2 in GH61s, Figure 3).²¹ As with GH61, the N-terminalactive site is at the center of an extended flat surface (ca. 30 ×40 Å), which is formed by the helical bundle and the narrow endof the β-sandwich. This surface presumably interacts in a face-to-facemanner with the surface of the substrate, bringing the copper intoclose contact with the polysaccharide.

Figure 3

Structural representationof Cu-BaCBM33, showingcopper active site on binding face.

Structural variation between the active sites of the apo and both copper-bound forms is minimal, revealing adegree of preorganizationfor copper binding (Figure S3), consistentwith the high copper-binding affinity. In the copper-bound form atthe N-terminal histidine active site, well-defined electron densityconsistent with a single, fully occupied copper ion was found, coordinatedin a T-shaped configuration of the histidine brace by the nitrogenatoms of two histidine side chains and the amino terminus [∠N_his–Cu–NH₂ = 96° and ∠NH₂–Cu–N_his = 98°], with Cu–N_his distances of 2.0 and 2.0 Å and a longer Cu–N(amino terminus) distance of 2.3 Å (Figure 4). Unlike GH61, there is no tyrosine/ate present in the apical positionof the coordination sphere, which is replaced by a phenylalanine inCBM33. Additionally, in contrast to GH61, the N-terminal histidineis not τ-N-methylated. These differences, if indeed presentin the wild-type enzyme, would amount to a significant variance inthe active site electronics of the copper site in CBM33 and pointtoward differing mechanisms of action between the two enzymes, possiblydictated by the oxidation requirements of different natural substrates.

Figure 4

Top: Electrondensity map contoured at 1σ in the active siteof Cu-BaCBM33; see Figure S4 for stereo view. Bottom: Line diagram of active site; cf., Figure 1 for comparison to active site of GH61.

Surprisingly, no further significant electron density(>0.3 σ)was found in the primary coordination sphere of the putative copper(II)ion, inconsistent with the dearth of known small molecule T-shapedcopper(II) complexes. Indeed, only one genuine copper(II) complexin a N₃ T-shaped geometry is known, and in this complexthe EPR spin Hamiltonian values are markedly different from thoseof Cu(II)-BaCBM33 (see below for further discussion).^31,32 The T-shaped coordination geometry in the structure of Cu-BaCBM33 is, however, consistent with a copper(I) formulation,especially because there is no difference in copper coordination spherewhen the crystals are soaked with sodium ascorbate and the structureredetermined. The possibility of a reduced copper oxidation stateis corroborated by small molecule studies of copper-bis(pyrazolyl)amine ligands, where the nitrogen atoms of the ligand occupy a T-shapedconfiguration, and studies of three coordinate copper in histidylhistidinecomplexes, both of which also have high redox potentials.^33,34

X-ray Absorption Spectroscopy Studies of Copper Oxidation State

To confirm the presence of copper(I) in the structures, we performeda Cu K-edge XANES study both of the in situ crystal during diffractiondata collection and of an aqueous solution of Cu(II)BaCBM33 at pH 5 both before and after exposure to synchrotron X-rays(Figure 5). X-ray exposure of a 0.6 mM solutionof Cu(II)-BaCBM33 at pH 5 led to the rapid (aftera single scan) formation of a pre-edge feature at 8982–3 eV.This feature could also be generated from the addition of a solutionof reducing agent (sodium ascorbate at pH 5), which is further knownto afford an EPR silent species. The XANES absorption at 8983 eV isassigned to a 1s to nonbonded 4p transition of a coordinatively unsaturatedcopper(I) ion; this transition is a reliable marker of copper oxidationstate and also of low (<4) coordination number.³⁴⁻³⁷ Moreover, in this case, the profileof the pre-edge region matches closely to that of a known small moleculecopper(I) complex in which the copper ion is coordinated in T-shapedconfiguration formed by two trans pyrazolyls and one bridgehead amine,^38,39 directly analogous to the observed copper structure in Cu-BaCBM33.

Figure 5

Left: 0.6 mM Cu-BaCBM33 solution at pH5 (1 eVsteps). Right: In crystallo (0.5 eV steps), successive Cu K-edge XANESfluorescence spectra (black line followed by red line) showing growthof pre-edge peak at 8982–3 eV. Blue spectrum (offset in ordinatefor clarity) is that of Cu-BaCBM33 solution/crystalpretreated with sodium ascorbate solution.

To link the crystallographic and solution phase studies,we performedfluorescence XANES studies of the in situ crystal. Notwithstandingtheir relatively low signal-to-noise, these spectra display the samepre-edge feature and behavior as that observed in solution. We thereforeinfer that the BaCBM33 copper-bound structure isof the copper(I) form, where the copper ion has been unavoidably photoreducedby the X-rays. This observation underlines the need for very considerablecaution in the interpretation of oxidation state and coordinationgeometry in the X-ray structures of copper enzymes that contain thecopper histidine brace structure, a point convincingly made some yearsago by Somerhalter et al.⁴⁰ and a featureof the early structures of CBM33 and GH61, which goes some way toexplain the confusion about the identity of the metal ion.^10,11

Redox Studies of CuBaCBM33

The ostensiblyhigh redox potential of CuBaCBM33 implied from theXAS studies was investigated using the method of titration of Cu(II)-BaCBM33 against a range of redox-active dyes with knownredox potentials, indicating a redox potential for CuBaCBM33 of between ∼275 and 370 mV (FigureS7). Recent studies of a Cu-CBP21 yielded a comparable valueof the redox couple of ca. 275 mV at pH 6 (vs SHE).²⁰

The relatively high potential is expected from thelow coordination number of the copper ion in CBM33. This has alsobeen observed in studies of copper histidylhistidine complexes.³⁴ Furthermore, low coordinate copper(I) centershave been proposed to be the sites of reactive-oxygen-species (ROS)production in copper amyloid-β fragments.³⁶ In both cases, the redox potential of the copper(I) complexand its reactivity toward dioxygen is critically dependent on thebalance of three coordinate versus two coordinate copper(I), which,in the context of GH61 and CBM33, implies a significant role in oxygenactivation for the central amino terminus in the coordination sphereof the copper ion. Additionally, the principal ROS product in copperamyloid-β fragments appears to be peroxide rather than superoxide,requiring the presence of a redox-active tyrosine/ate within the peptidefragment. A similar situation could occur within GH61 and CBM33, bothof which have conserved tyrosines or tryptophans within their sequences(see below).^19,21

X-Band EPR Spectroscopyof met-CuBaCBM33 and Azide BindingStudies

In the absence of structuralinformation from X-ray crystallography, the nature of the copper(II)site in Cu(II)-BaCBM33 was investigated using frozensolution X-band EPR spectroscopy at 155 K. Cu(II)-BaCBM33 exhibits a rhombic spectral envelope (g_x ≠ g_y ≠ g_z) but where the SOMO has significant d(x²–y²) character, indicatinga mononuclear copper(II) ion in a single binding site (Figure 6) with distorted-axial coordination geometry. Simulationof the spectrum was hampered by the second-order nature of the perpendicularregion, making a determination of the g_x and g_y and |A_x| and |A_y| tensor values unreliable.Spin Hamiltonian tensor values in the parallel direction, however,could be modeled accurately with g_z = 2.25 and |A_z| = 125 G (0.0135 cm^–1). These values place Cu(II)-BaCBM33 between the usual Peisach–Blumberg classificationsof type 1 and type 2 copper enzymes, although the overall axial envelopeof the EPR signal would suggest that a type 2 classification is appropriate.²⁹ The somewhat low |A_z| value in combination with low g_z could arise from increased metal–ligandcovalency or, more likely given the rhombicity of the spectrum andthe potential absence of ligands on the z-axis (seestructure discussion below), a coordination geometry at the copperthat is distorted away from a local axial symmetry, possibly by thecoordination of one or two water molecules, neither of which is directlytrans to the amino terminus. This distortion allows the SOMO to mixwith 3d(z²) and/or 4s orbitals, affordinga greater relative contribution of spin-dipolar and/or Fermi contactterms to the hyperfine coupling.^41,42 Additionalevidence for a distorted coordination geometry comes from noting thesimilarity of the g_z and|A_z| values for Cu(II)-BaCBM33 with those of the copper site in Cu–Zn superoxidedismutase, in which the copper has a distorted square planar coordinationgeometry.⁴³ Furthermore, an existing X-raystructure of a CBP21, which, despite the incorrect assignment of themetal ion as Na⁺ at the active site (PDB entry 2BEM), exhibits a distortedsquare planar coordination at the metal where the three nitrogen atomsof the histidine brace and the metal ion occupy a plane and wherea water molecule in the fourth coordination site is significantlydeviated from this plane.¹¹

Figure 6

X-Band (9 GHz, 155 K)EPR spectrum (top) with simulation in red(bottom) of Cu(II)BaCBM33 at pH 5 (15% v/v glycerol).

Addition of sodium azide to asolution of Cu(II)BaCBM33 at pH 5 affords changesin both UV/vis and EPR spectra. Inthe latter, addition of stoichiometric azide gives a shift in g_z to 2.23 and |A_z| to 0.0125 cm^–1,but no change in the overall spectral envelope. Addition of a 10-foldexcess of azide affords a significant change with the formation ofnew peaks but with loss of some of the fine structure (Figure S5), indicative of coordination to thecopper(II) center. In the UV/vis spectrum, a single intense band (ca.900 dm³ mol^–1 cm^–1)appears at ∼390 nm assignable to an azide to copper chargetransfer band at a mononuclear copper site.⁴⁴ The appearance of this band is accompanied by a small increase inthe intensity of the d–d transition absorption of the Cu(II)center at ca. 690 nm (Figure 6).

Thereare distinct differences in the EPR spectrum of Cu(II)-BaCBM33 and that of Cu(II)-TaGH61. In thelatter, the g_z and |A_z| values of 2.27 and 0.0162cm^–1 place GH61 squarely within the type 2 classificationof copper site and are also indicative of a copper(II) ion withinan axial coordination geometry. This geometry was confirmed by thesubsequent X-ray diffraction studies of GH61. It is currently unclearwhy there are such differences in the coordination geometries of the met forms of both CBM33 and GH61, but the difference islikely to be of functional significance given the differences in thecopper active site structures of CBM33 and GH61 (see below).

SequenceConservation Analysis of CBM33s and Comparison to CBP21and GH61s

An amino acid sequence alignment of BaCBM33 with a range of selected CBM33s is shown in Figure S6. Outside the active site histidines, strict conservationis observed in parts of the β-sandwich and in a collection oftryptophan residues adjacent to the active site, where the latteris probably associated with an electron transfer function. This particularconservation of tryptophans is also observed in CBP21 chitin oxidases,as previously noted by Vaaje-Kolstad et al.¹⁹

Figure 7

Activesite conserved residues in CBM33 depicting the occlusionof Cu(II) axial coordination sites by alanine 123 and phenylalanine196. Sections of the sequence alignment with two other CBM33s (B.a= Bacillus amyloliquefaciens, S.m = Serratia marcescens, and E.f = Enterococcusfaecalis)^19,23 are shown surroundingeach of the active site residues, which are shown in bold. Their associatedpercentage identities from the full sequence alignment shown in Figure S6 are indicated by the histograms belowthe sequences according to the key.

An unusual feature in CBM33 is observed in the strict conservationof an alanine residue at position 123 at the end of a β strand.The methyl group of this alanine is close to the apical coordinationsite on the copper ion (Cu···C = 3.9 Å), therebyproviding a degree of steric congestion at this coordination site.What makes this feature notable is that the opposite apical site isoccupied by a highly conserved phenylalanine side chain (position196), which has a near “end-on” aspect to the copperion with a closest Cu···C distance of 3.7 Å (Figure 7). The combination of the alanine and phenylalaninesuggests that coordination to the copper in CBM33 is restricted toequatorial sites only (Figure 7). This is insharp contrast to GH61 where the apical sites are occupied by theoxygen atoms of a tyrosine/ate and a water molecule, where the watermolecule could be replaced by either substrate or an oxygen molecule,as suggested by Li et al.²¹ Whatever thereasons for the differences between CBM33 and GH61, the contrastingarrangement of amino acid side chains in the copper ions’ secondarycoordination spheres, including that of the τ-N-methylated N-terminalhistidine in GH61, is indicative of different mechanisms of actionbetween the two enzymes.

Figure 8

Binding face of BaCBM33 indicatingconserved residues.Region colored red is copper active site, which is surrounded by highlyconserved regions consisting of residues capable of forming hydrogenbonds with substrate. L2 loop region is to bottom right of figure,showing no noticeable conservation, in contrast to type 2 PMOs (GH61s),which exhibit conservation of some aromatic residues.²¹

In a further indication of alternativemechanisms, the conservedresidues on the binding face of CBM33 differ from that of GH61s. Inthe former, the principal conservation pattern is found in amino acidside chains, which can engage in hydrogen-bond interactions with apolysaccharide substrate. These residues lie in two areas immediatelyadjacent to the copper active site. The two regions lie on oppositesides of the copper ion, thus providing an environment that likelydirects a specific orientation of the substrate with respect to thecopper and thus any reactive oxygen species generated at the activesite. Such a directed interaction with the substrate is in accordancewith the observation that CBM33s react principally at the C1 siteof β-linked polysaccharides; it may also offer an explanationas to why only even-numbered oligosaccharides are produced by CBM33insofar as the up–down–up–down orientation patternof individual glycoside units within chitin and cellulose will onlyproject C–H bonds (from C–1) toward the enzyme bindingface every other glycosidic unit, as first suggested by Vaaje-Kolstadet al.^19,23

GH61s appear to bind to substratethrough multiple aromatic–carbohydrateinteractions. This was shown by Li et al., who identified conservedtyrosine residues on the α-helical L2 loop in polysaccharideoxygenases and also on residues near the copper active site (Figure 8).²¹ The residues arenot only in different positions on the binding face when comparedto CBM33s, but also probably interact with the cellulose substratein a somewhat less directional way than the potential hydrogen-bondinginteractions seen in CBM33. This is commensurate with a wider rangeof oxidation sites on cellulose that is observed with GH61 oxidativeaction, especially type 3 PMOs,²¹ and thatoxidation does not only occur at the exposed C1–H bonds oncellulose, as appears to be the case in CBM33.

It is not possibleto be more definitive about the conservationdifferences between CBM33 and GH61 because we are unable to rule outthe possibility of post-translational modifications on CBM33, whichcould introduce extra functionality at the active site, for instance,oxidation of phenylalanine to tyrosine and potential methylation ofthe N-terminal histidine. Indeed, we observe here that one CBM33 sequencenaturally has a tyrosine in place of phenylalanine 196. Nor can wecompletely rule out a coordinative role for a highly conserved tyrosineat position 197, although note that this appears to be unlikely giventhe large structural disruption this would entail and the absenceof any conformational flexibility in this region as observed acrossseveral sets of deposited coordinates. Additionally, an interrogationof the CAZy database shows that GH61s can appear as multidomain proteinsin which the oxygenase domain is fused to other known cellulose-bindingmodules (e.g., CBM1) through which binding to cellulose will be significantlyaffected.⁴⁵ Similarly, CBM33 enzymes areoften appended to diverse other protein modules involved in hydrolysisand binding of carbohydrates, as reviewed recently by Horn et al.⁴ Notwithstanding these caveats, however, a structuralbasis for the known differences in mechanisms of action of CBM33sand GH61s does emerge without the need to introduce significant post-translationalmodifications in CBM33s, and may well therefore be a reliable basisfor understanding differences in reactivity.

Expressionand Purification of BaCBM33

The coding sequenceof Bacillus amyloliquefaciens CBM33(NCBI Reference Sequence: NC_014551.1) from nucleotide 82 to 621was codon optimized for expression in Escherichia coli, synthesized with a pelB leader sequence to direct the protein tothe periplasm, and cloned into the pET-11a vector, by GenScript. Theresulting pET11a-BaCBM33 construct was used to transform BL21 (DE3) E. coli, which were grown from a single colony insix 500 mL LB cultures to an A₆₀₀ of 0.4at 37 °C shaking at 180 rpm. The temperature was then reducedto 16 °C, and the cells were allowed to grow further to an A₆₀₀ of 0.6–0.8 when expression was inducedby the addition of IPTG to a final concentration of 1 mM. The cultureswere left overnight before the cells were harvested by centrifugationat 11 000g for 30 min at 4 °C.

Cells were immediately resuspended in 5 volumes of ice cold resuspensionbuffer (50 mM Tris pH 8.0, 200 mM NaCl, 20% w/v sucrose). To isolatethe periplasmic fraction, 40 μg of hen egg white lysozyme (Sigma-Aldrich)was added for every gram of cell paste and left on ice for 1 h. 60μL of 1 M MgSO₄ was then added per gram of cell paste,and the suspension was incubated for a further 20 min on ice. Celldebris was then removed by centrifugation at 10 000g, 4 °C for 10 min, and the supernatant containingthe periplasmic fraction was removed into a fresh tube. This was diluted4 fold with buffer A (50 mM Tris pH 8.0) and passed through a 5 mLHiTrap Q Sepharose column equilibrated in the same buffer collectingthe flow through. The column was then stripped with 100% buffer B(50 mM Tris pH 8.0, 2 M NaCl). BaCBM33 did not bind to the columnand was present in the flow through, which was precipitated by theaddition of solid ammonium sulfate to a final concentration of 3.5M and left stirring overnight at 4 °C. The protein was then isolatedby centrifugation at 38 000g for 30 min, thesupernatant was discarded, and the pellet was dissolved in 10 volumesof buffer C (20 mM sodium acetate pH 5.0, 250 mM NaCl, 5 mM EDTA).The protein was then concentrated to less than 10 mL and applied toa HiLoad 26/60 Superdex 75 column equilibrated in GF buffer (20 mMsodium acetate pH 5.0, 250 mM NaCl). Peak fractions containing pureBaCBM33 were pooled and concentrated using a Sartorius 10 kDa cutoff concentrator at 4000g. Protein concentrationsfor all subsequent experiments were determined by measuring the A₂₈₀ using an extinction coefficient of 44 920M^–1 cm^–1 and a molecular weightof 19 821 Da.

Redox Potential Measurements

Theredox indicator dyesthionine, tetramethylphenylenediamine (TMP), and 2,6-dichloro-phenolindophenol(DCPIP) were purchased from Sigma. A three-electrode setup was usedto determine accurately the reduction potential of the redox indicatorsand potassium ferricyanide in 100 mM sodium acetate buffer, pH 5.A stationary Pt disc working electrode (BASi), a Pt wire counter electrode,and a saturated calomel reference electrode (SLS) were all placedinto an all-glass electrochemical cell. Cyclic voltammograms (50 mVs^–1) were measured for 1 mM solutions of each ofthe redox compounds under a flow of Ar. A correction factor of E_SHE= E_SCE + 0.241V was used. The resultant scans are shown in FigureS7. The anodic and cathodic peak potentials, as determinedby the electrochemical software (Epsilon BASi), were averaged to givethe midpoint reduction potentials, summarized in Table S2. To judge if the dye could reduce BaCBM33, up to 10 μL of approximately 0.5 mM enzyme was injectedinto a suba-sealed cuvette containing 150 μL of reduced dyesolution (absorbance <0.2 AU) and a small stirrer bar. Following2 min of stirring, any resulting color changes were monitored usingUV–vis spectroscopy. Control experiments were conducted withinjection of buffer instead of enzyme, and it was confirmed that CBM33was required to oxidize thionine or TMP in the cuvette.

EPR Spectroscopy

Continuous wave X-band frozen solutionEPR spectra of 0.5 mM solution of Cu(II)-BaCBM33 (15% v/v glycerol)at pH 5 (acetate buffer) and 155 K were acquired on a Bruker ESP 300spectrometer operating at 9.072 or 9.35 GHz, with a modulation amplitudeof 4G and microwave power of 5 mW. Spectra were referenced againstDPPH. Spectral simulation was carried out using Easyspin 4.0.0 ona desktop PC. Simulation parameters were as follows: g_x = 2.075, g_y = 2.086, g_z = 2.255; |A_x|= 39, |A_y| = 90, |A_z| = 125 G. Strain: A_xx = 210, A_yy = 210, A_zz = 210 G. Linewidth: 40 G (Gaussian), 40 G (Lorentzian). g_z and |A_z| were determined accurately from the twoabsorptions at low field where it was assumed that these absorptionswere separated from other aspects of the absorption envelope. It wasassumed that g and A tensors wereaxially coincident. Accurate determination of the g_x, g_y, |A_x|, and |A_y| was not possible due tothe second-order nature of the perpendicular region and the significantlevel of conformational heterogeneity, although it was noted thatsatisfactory simulation could only be achieved with one particularset of spin Hamiltonian values.

Solution XANES Spectroscopy

XANES spectroscopy datawere collected on a 0.6 mM solution of Cu(II)-BaCBM33 at pH 5, whichhad been flash-frozen to 77 K (0.7 mM BaCBM33 was prepared in 20 mMsodium acetate pH 5.0, 250 mM NaCl to which Cu(NO₃)₂·3H₂O was added to a final concentration of0.6 mM; to produce a Cu(I) sample, ascorbate was added to a finalconcentration of 10 mM). Data were acquired on the sample at 90 Kat the B18 Core Spectroscopy beamline at Diamond Light Source, Oxfordshire,UK. At the time of the measurement, the Diamond synchrotron was operatingat a ring energy of 3 GeV in a 10 min top-up mode for a ring currentof 301 mA. The beamline was equipped with a Si(111) double crystalmonochromator, and harmonic rejection was achieved through the useof two Pt-coated mirrors operating at an incidence angle of 9 mrad.The monochromator was calibrated using the first maximum in the derivativein the edge region of the XAS spectra of a copper foil placed betweenthe second and third ion chambers at 8979 eV. Estimated flux of beamat 8 keV = 5 × 10¹¹ ph/s. Data were collected in fluorescencefrom 8779 to 9020 eV using a nine-channel Ge solid-state detectorat the copper K absorption edge (∼8980 eV) in 1 eV steps. Thesample was contained in a 5 mm light path PTFE 400 μL cell with25 μm thick windows made from Kapton foil. The measurementswere made at 90 K. The incident beam intensity was measured usinga 30 cm ion chamber optimized using a helium–nitrogen gas mixtureto absorb 30% and 70% of the beam in I0, respectively.

XANES DataCollection in the Crystal

X-ray fluorescencescans were performed on crystals, which had not previously been exposedto X-rays on station I03 of Diamond Light Source, using a Vortex multicathode,single element detector. Crystals from copper cocrystallizations,as grown for the structure determination, were washed through threedrops of cryo-protectant solution to remove excess copper prior toplunging in liquid nitrogen. To produce Cu(I) control samples, 20mM ascorbate was included in the cryo-protectant soaking solution.Fluorescence scans were performed across the copper K-edge from 8942to 9017 eV with step sizes of 5 eV from 8942 to 8962 eV, 0.5 eV acrossthe edge from 8962 to 8997 eV, and 5 eV from 8997 to 9017 eV.

IsothermalTitration Calorimetry

Isothermal titrationcalorimetry was performed using a VP-ITC calorimeter (MicroCal). Typicallyprotein was present in the cell between 10 and 120 μM with a10-fold more concentrated solution of CuCl₂ in the syringe.Titrations were initially performed at 283 K, but the same resultscould later be obtained at 298 K also. After an initial 2 μLinjection, which is discarded in the data analysis, 10 μL injectionswere used during the titration with a 5 min interval between eachinjection at pH 5.0 and 6.0 and 8 min intervals at pH 7.0 where bindingwas slower. The buffers used during the titrations were either 20mM sodium acetate pH 5.0, 250 mM NaCl, 20 mM Bis-Tris pH 6.0, 250mM NaCl, or 20 mM Bis-Tris pH 7.0, 250 mM NaCl with the CuCl₂ solution prepared in exactly the same buffer. All data were analyzedusing the Origin 7 software package (MicroCal). Heats of dilutionwere subtracted from the data, but the analysis routinely returneda substoichiometric binding of Cu. Modification of the injection systemto remove Cu-containing brass components improved the stoichiometries,but these rarely exceeded 0.86. This is likely due to a portion ofthe protein having acquired Cu²⁺ given the very high affinityof the interaction from the glassware. To give meaningful ΔH values (the K_Ds are unaffected),the protein concentration was therefore adjusted in the software toreflect the 1:1 stoichiometry of copper to protein seen in the crystalstructure.

Differential Scanning Fluorimetry

Differential scanningfluorimetry was used to determine the melting temperature of BaCBM33 with and without metal ions. Stability measurementswere performed using an Agilent MX3000P QPCR machine and the fluorescentdye SYPRO orange (Sigma-Aldrich) diluted 2000 fold from the stocksolution. Fluorescence was measured with excitation and emission wavelengthsof 517 and 585 nm, respectively. All experiments were performed in20 mM sodium acetate pH 5, 250 mM NaCl with protein at 25 μM,and a total volume of 30 μL. Fluorescence was monitored whileincreasing the temperature in steps of 1 °C at 30 s intervalsfrom 25 to 96 °C. The fluorescence was significantly quenchedin the presence of copper, but a melting curve could still be obtained.Melting temperatures (T_m) were calculatedby fitting a sigmoidal curve to the data using the MTSA⁴⁶ program for MATLAB.

Crystallization of Apo and Cu-Bound BaCBM33

To form the Cu complex,Cu(NO₃)₂·3H₂O was added to theprotein to a final concentration of 1 mM. Apo andCu cocrystallization trials were then set up inparallel with a protein concentration of 7 mg/mL using a Mosquitorobot (TTP Labtech). The best crystals in both cases were obtainedin condition D1 of the PACT screen (Qiagen): 0.1 M MMT buffer pH 4.0,25% PEG-1500. Crystals were used directly from these initial screensfor subsequent data collection. Crystals used to determine the Cu-bound/ascorbatesoaked structure were cocrystallized with copper in the same way asdescribed above.

Diffraction Data Collection, Processing,and Structure Determination

Crystals were cryo-cooled fordata collection by first soakingfor 30 s in mother liquor supplemented with 20% v/v ethylene glycolbefore plunging directly in liquid nitrogen. For the ascorbate soak,20 mM sodium ascorbate was included in the cryo-protectant. Diffractiondata were collected at Diamond Light Source, beamlines I04-1 and I03at wavelengths of 0.917 and 0.976 Å, respectively. Data wereindexed and integrated using XDS⁴⁷ withsubsequent processing performed using the CCP4 software package.⁴⁸ The Serratia marcescens CBP21 structure (pdb 2ben)⁴⁹ was prepared as a molecularreplacement search model using CHAINSAW,⁵⁰ cutting back side chains to their nearest common atom. The Apo and Cu-bound structures were initially determined bymolecular replacement using PHASER,⁵¹ bothcontaining two molecules in the asymmetric unit. Following structuresolution, pseudotranslational symmetry was detected in the apo-BaCBM33 data. The structure was therefore solved againusing MOLREP inputting the pseudo translation vector 0.500, 0.000,0.127 for the off-origin Patterson peak. ARPwARP⁵² was then used to rebuild the initial models before subsequentmanual building and refinement using COOT⁵³ and REFMAC5,⁵⁴ respectively. Local NCSrestraints were applied using the automatic NCS option in REFMAC5.The ascorbate soaked crystals were isomorphous to the copper-freecrystals, and so the copper-free structure was simply refined againstthese data, with waters and flexible loops removed, to yield the structure.This was rebuilt and refined with COOT and REFMAC5 as for the others.All data processing and refinement statistics can be found in Table S1. Coordinates and accompanying structurefactors for the apo-, Cu-bound, and Cu-bound/ascorbatesoaked enzyme have been deposited in the protein data bank with accessioncodes 2YOW, 2YOX, and 2YOY, respectively.