controlled degradation 
 of abundant biomass is a sine qua non for 
 the future success of bioethanol production . in this regard , finding a means of overcoming the 
 recalcitrance of cellulosic , lignocellulosic , or chitinotic materials 
 either by chemical or by enzymatic methods is a major objective . of 
 these methods enzymatic solutions offer promise especially as recent 
 months have seen strides toward a fuller appreciation of the consortia 
 of ligninases , cellulases , and chitinases deployed by saprophytes 
 and heterotrophs in the degradation of biomass . in this context , an important recent advance was the discovery of 
 a class of enzymatic oxidases , and their action on polysaccharides , 
 reported by harris et al . and vaaje - kolstad et al . , who demonstrated 
 that effective fungal and bacterial depolymerization of polysaccharides 
 hinges upon the initial action of structure - disrupting enzymes classified as gh61 and cbm33 , respectively . subsequently , 
 it was shown that gh61s from thermoascus aurantiacus and thielavia terrestris are new 
 types of copper - dependent oxidases with an unusual active site , where 
 the copper ion is bound by two histidines of the so - called histidine 
 brace ( figure 1 ) . the active site is notable for its -n - methylated n - terminal 
 histidine , the functional requirement of which is unclear . it is also 
 similar to part of the copper active site of particulate methane monoxygenase , 
 another powerful copper oxidase . in contrast to gh61 , however , and despite their potential for biofuel 
 production , the structural and mechanistic details of cbm33 enzymes 
 are less certain . while initial structural reports suggested that na , zn , or mg might be the metal at the active site , these have now been 
 shown to be incorrect as the activity of a bacterial cbm33 from enterococcus faecalis(18,19 ) was recently 
 demonstrated also to be copper - dependent . site - directed mutagenesis of the coordinated tyrosine / ate leads to 
 reduction in activity , while mutation of glutamine leads to inactivation . a recent nmr structure 
 ( pdb 2lhs ) of 
 a cbp21 cbm33 showed that the overall 
 solution structure of cbp21 is similar to that of the crystallized 
 enzyme , but while cu perturbs the histidine nmr signals ( allowing 
 pka determination ) showing the expected 
 coordination of copper at the active site , no details of the copper 
 coordination geometry could be determined from this study . also awaiting fuller elucidation is an understanding 
 of the difference in mechanisms of action of cbm33 and gh61 . for instance , 
 analysis of the degradation products from the action of gh61 indicates 
 oxidation at c1 , c4 , or c6 of the glycosidic unit , dependent on the 
 subclass of gh61 carrying out the oxidation . in these cases , gh61 action affords a linear sequence of oligosaccharide 
 products with degrees of polymerization ( dp ) = 2,3,4,5,etc . in contrast , where studied , cbm33 
 oxidation appears only to occur at c1 and affords principally oligosaccharides 
 with dp = 2,4,6,etc . from a structural perspective , 
 both enzyme classes exhibit an overall 
 structure with a beta - sandwich core , which lies roughly perpendicular 
 to an extended flat face , the center of which contains the active 
 site . for gh61 , substrate - binding at the face is thought to be mediated 
 mainly by aromatic  carbohydrate interactions . in cbm33 , a lack of aromatic amino acid side chains at the binding 
 site suggests that binding to polysaccharides is mediated through 
 different interactions exemplified by proposals for the interaction 
 of the have a functional requirement 
 for a reducing 
 equivalent , either a sugar dehydrogenase exemplified by cellobiose 
 dehydrogenase or a small molecule reductant , which , along with the need for dioxygen , is 
 commensurate with a catalytic cycle in which dioxygen activation at 
 a reduced copper site is a key step . indeed , through further structural 
 studies of an oxygenated form of a cu - gh61 and in parallel with other 
 known copper oxygenases such as peptidyl glycine monoxygenase , marletta et al . speculated on a reaction sequence 
 for gh61 in which , following the formation of a copper(i ) state and 
 addition of dioxygen , copper(ii)-superoxide is a potential oxidative 
 species . this proposal is based on a structure of 
 gh61 , which shows the copper ion coordinated in an axially elongated 
 geometry , with the equatorial plane occupied by the three nitrogen 
 atoms of the histidine brace . a water molecule occupies one axial 
 position , and the other axial position has electron density that is 
 assigned to a superoxide with a cuo distance of 
 2.9  . notwithstanding this structure , 
 in - depth mechanistic studies are urgently required to examine not 
 only the proposal of a copper - superoxide but also , and perhaps more 
 importantly , whether other types of reactive copper  oxygen 
 species could be formed as part of the reaction cycle . as part 
 of the continuing drive toward understanding the mechanisms 
 of action of gh61s and cbm33s , we report here the isolation , characterization , 
 and full three - dimensional x - ray structures of the apo and copper - bound forms of a cbm33 from bacillus amyloliquefaciens . as initially demonstrated by aachmann et al . , we confirm that copper(ii ) 
 binding to the apo enzyme is very tight indeed , and 
 we further show that copper binding is accompanied by a substantial 
 increase in protein stability . additionally , 
 using epr spectroscopy , we reveal details of the copper coordination 
 sphere , which , in contrast to gh61 , shows the copper ion in a coordination 
 geometry significantly distorted from axial , such that it lies somewhat 
 between the usual peisach  blumberg type 1 and type 2 classifications . three - dimensional structure and x - ray absorption 
 spectroscopy studies of the reduced form display a copper(i ) ion in 
 a t - shaped coordination geometry , where the copper has been photoreduced 
 by the incident x - rays . the bacillus 
 amyloliquefaciens cbm33 
 domain , ( first described as chitin - binding protein chbb from a chitinolytic 
 strain of b. amyloliquefaciens ; hereafter bacbm33 ) , was produced 
 from expression of the codon - optimized gene and subsequent periplasmic 
 secretion in e. coli allowing processing 
 of the signal peptide to yield the free n - terminal histidine . standard 
 purification with q sepharose and superdex columns yielded good quantities 
 of 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 kd of 6 nm at ph 5 ( c value 
 > 1000 ) , which is comparable to the value of 55 nm recently found 
 for 
 the binding of cu(ii ) to cbp21 . copper(ii ) 
 affinity is lower at higher ph giving kd values of 40 nm at ph 6 ( c value = 325 ) and 80 
 nm at ph 7 ( c = 435 ) , with a copper to protein stoichiometry 
 of approximately 0.8:1 ( figure 2 ) . the small 
 increase in kd with ph is possibly due 
 to competition for copper binding from the buffers used at higher 
 ph or potentially from the deprotonation of active site residues involved 
 in the stabilization of a metal - bound water molecule ( see below for 
 further discussion ) , although the reasons for these differences await 
 a more detailed binding study . in either case , isothermal calorimetry of metal binding 
 to bacbm33 : 
 ( a ) cu(ii ) at ph 6 ( see figure s1 for other 
 ph measurements ) , ( b ) zn(ii ) at ph 5 . binding of nickel(ii ) , manganese(ii ) , and zinc(ii ) at ph 
 5 was 
 also investigated using itc , with zn(ii ) the only metal showing any 
 measurable binding ( figure 2 ) . to corroborate 
 itc data , the thermostability of the protein was measured in the presence 
 of these metals . in the presence of cu(ii ) , the melting temperature 
 ( tm ) increased by 20 k ( figure s2 ) , while in the presence of ni(ii ) and zn(ii ) , a 
 less significant shift of 7 k was observed reflecting the lower affinity 
 of cbm33 for these metals . the potential binding 
 of manganese ions was of interest because there is a single report 
 of gh61 using mn as a metal ion in the absence of copper . however , in the case of bacbm33 , 
 no mn binding could be detected , strongly suggesting that cbm33s are 
 uniquely copper enzymes . we also note that the enzyme does not bind 
 further copper ions with any significant binding constant , analogous 
 to gh61s and commensurate with the active site in cbm33s being a mononuclear 
 copper active site , and in contrast to the dicopper site proposed 
 for copper methane monoxygenase in which the histidine brace also 
 appears . such selective binding of a single copper corroborates the 
 recent reports from activity assays that cbm33s should be classified 
 as mononuclear copper - dependent oxygenases . to investigate 
 the nature of the structure of bacbm33 in more detail 
 and , in particular , the copper site , the three - dimensional 
 structures of bacbm33 in apo , copper - bound , 
 and copper - bound / ascorbate - soaked forms were solved by molecular replacement 
 at resolutions of 1.8 , 1.9 , and 1.7  , respectively . the structures 
 are analogous to those already reported for cbm33 and gh61 consisting 
 of a core immunoglobin - like -sandwich domain with an adjacent 
 helical bundle ( defined as loop l2 in gh61s , figure 3 ) . as with gh61 , the n - terminal 
 active site is at the center of an extended flat surface ( ca . 30  
 40  ) , which is formed by the helical bundle and the narrow end 
 of the -sandwich . this surface presumably interacts in a face - to - face 
 manner with the surface of the substrate , bringing the copper into 
 close contact with the polysaccharide . structural representation 
 of cu - bacbm33 , showing 
 copper active site on binding face . structural variation between the active sites of the apo and both copper - bound forms is minimal , revealing a 
 degree of preorganization 
 for copper binding ( figure s3 ) , consistent 
 with the high copper - binding affinity . in the copper - bound form at 
 the n - terminal histidine active site , well - defined electron density 
 consistent with a single , fully occupied copper ion was found , coordinated 
 in a t - shaped configuration of the histidine brace by the nitrogen 
 atoms of two histidine side chains and the amino terminus [ nhis  cu n 
 ( amino terminus ) distance of 2.3  ( figure 4 ) . unlike gh61 , there is no tyrosine / ate present in the apical position 
 of the coordination sphere , which is replaced by a phenylalanine in 
 cbm33 . additionally , in contrast to gh61 , the n - terminal histidine 
 is not -n - methylated . these differences , if indeed present 
 in the wild - type enzyme , would amount to a significant variance in 
 the active site electronics of the copper site in cbm33 and point 
 toward differing mechanisms of action between the two enzymes , possibly 
 dictated by the oxidation requirements of different natural substrates . top : electron 
 density map contoured at 1 in the active site 
 of cu - bacbm33 ; see figure s4 for stereo view . surprisingly , no further significant electron density 
 ( > 0.3  ) 
 was found in the primary coordination sphere of the putative copper(ii ) 
 ion , inconsistent with the dearth of known small molecule t - shaped 
 copper(ii ) complexes . indeed , only one genuine copper(ii ) complex 
 in a n3 t - shaped geometry is known , and in this complex 
 the epr spin hamiltonian values are markedly different from those 
 of cu(ii)-bacbm33 ( see below for further discussion ) . 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 sphere 
 when the crystals are soaked with sodium ascorbate and the structure 
 redetermined . the possibility of a reduced copper oxidation state 
 is corroborated by small molecule studies of copper - bis(pyrazolyl ) 
 amine ligands , where the nitrogen atoms of the ligand occupy a t - shaped 
 configuration , and studies of three coordinate copper in histidylhistidine 
 complexes , both of which also have high redox potentials . to confirm the presence of copper(i ) in the structures , we performed 
 a cu k - edge xanes study both of the in situ crystal during diffraction 
 data 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 solution 
 of cu(ii)-bacbm33 at ph 5 led to the rapid ( after 
 a single scan ) formation of a pre - edge feature at 89823 ev . this feature could also be generated from the addition of a solution 
 of reducing agent ( sodium ascorbate at ph 5 ) , which is further known 
 to afford an epr silent species . the xanes absorption at 8983 ev is 
 assigned to a 1s to nonbonded 4p transition of a coordinatively unsaturated 
 copper(i ) ion ; this transition is a reliable marker of copper oxidation 
 state and also of low ( < 4 ) coordination number . moreover , in this case , the profile 
 of the pre - edge region matches closely to that of a known small molecule 
 copper(i ) complex in which the copper ion is coordinated in t - shaped 
 configuration formed by two trans pyrazolyls and one bridgehead amine , directly analogous to the observed copper structure in cu - bacbm33 . left : 0.6 mm cu - bacbm33 solution at ph 
 5 ( 1 ev 
 steps ) . right : in crystallo ( 0.5 ev steps ) , successive cu k - edge xanes 
 fluorescence spectra ( black line followed by red line ) showing growth 
 of pre - edge peak at 89823 ev . blue spectrum ( offset in ordinate 
 for clarity ) is that of cu - bacbm33 solution / crystal 
 pretreated with sodium ascorbate solution . to link the crystallographic and solution phase studies notwithstanding 
 their relatively low signal - to - noise , these spectra display the same 
 pre - edge feature and behavior as that observed in solution . we therefore 
 infer that the bacbm33 copper - bound structure is 
 of the copper(i ) form , where the copper ion has been unavoidably photoreduced 
 by the x - rays . this observation underlines the need for very considerable 
 caution in the interpretation of oxidation state and coordination 
 geometry in the x - ray structures of copper enzymes that contain the 
 copper histidine brace structure , a point convincingly made some years 
 ago by somerhalter et al . and a feature 
 of the early structures of cbm33 and gh61 , which goes some way to 
 explain the confusion about the identity of the metal ion . the ostensibly 
 high redox potential of cubacbm33 implied from the 
 xas studies was investigated using the method of titration of cu(ii)-bacbm33 against a range of redox - active dyes with known 
 redox potentials , indicating a redox potential for cubacbm33 of between 275 and 370 mv ( figure 
 s7 ) . recent studies of a cu - cbp21 yielded a comparable value 
 of the redox couple of ca . the relatively high potential is expected from the 
 low coordination number of the copper ion in cbm33 . furthermore , low coordinate copper(i ) centers 
 have been proposed to be the sites of reactive - oxygen - species ( ros ) 
 production in copper amyloid- fragments . in both cases , the redox potential of the copper(i ) complex 
 and its reactivity toward dioxygen is critically dependent on the 
 balance of three coordinate versus two coordinate copper(i ) , which , 
 in the context of gh61 and cbm33 , implies a significant role in oxygen 
 activation for the central amino terminus in the coordination sphere 
 of the copper ion . additionally , the principal ros product in copper 
 amyloid- fragments appears to be peroxide rather than superoxide , 
 requiring the presence of a redox - active tyrosine / ate within the peptide 
 fragment . a similar situation could occur within gh61 and cbm33 , both 
 of which have conserved tyrosines or tryptophans within their sequences 
 ( see below ) . in the absence of structural 
 information from x - ray crystallography , the nature of the copper(ii ) 
 site in cu(ii)-bacbm33 was investigated using frozen 
 solution x - band epr spectroscopy at 155 k. cu(ii)-bacbm33 exhibits a rhombic spectral envelope ( gx  gy  gz ) but where the somo has significant d(x y ) character , indicating 
 a mononuclear copper(ii ) ion in a single binding site ( figure 6 ) with distorted - axial coordination geometry . simulation 
 of the spectrum was hampered by the second - order nature of the perpendicular 
 region , making a determination of the gx and gy and |ax| and |ay| tensor values unreliable . spin hamiltonian tensor values in the parallel direction , however , 
 could be modeled accurately with gz = 2.25 and |az| = 125 g ( 0.0135 cm ) . blumberg classifications 
 of type 1 and type 2 copper enzymes , although the overall axial envelope 
 of the epr signal would suggest that a type 2 classification is appropriate . the somewhat low |az| value in combination with low gz could arise from increased metal ligand 
 covalency or , more likely given the rhombicity of the spectrum and 
 the potential absence of ligands on the z - axis ( see 
 structure discussion below ) , a coordination geometry at the copper 
 that is distorted away from a local axial symmetry , possibly by the 
 coordination of one or two water molecules , neither of which is directly 
 trans to the amino terminus . this distortion allows the somo to mix 
 with 3d(z ) and/or 4s orbitals , affording 
 a greater relative contribution of spin - dipolar and/or fermi contact 
 terms to the hyperfine coupling . additional 
 evidence for a distorted coordination geometry comes from noting the 
 similarity of the gz and 
 |az| values for cu(ii)-bacbm33 with those of the copper site in cu zn superoxide 
 dismutase , in which the copper has a distorted square planar coordination 
 geometry . furthermore , an existing x - ray 
 structure of a cbp21 , which , despite the incorrect assignment of the 
 metal ion as na at the active site ( pdb entry 2bem ) , exhibits a distorted 
 square planar coordination at the metal where the three nitrogen atoms 
 of the histidine brace and the metal ion occupy a plane and where 
 a water molecule in the fourth coordination site is significantly 
 deviated from this plane . 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 a 
 solution of cu(ii)bacbm33 at ph 5 affords changes 
 in both uv / vis and epr spectra . in 
 the latter , addition of stoichiometric azide gives a shift in gz to 2.23 and |az| to 0.0125 cm , 
 but no change in the overall spectral envelope . addition of a 10-fold 
 excess of azide affords a significant change with the formation of 
 new peaks but with loss of some of the fine structure ( figure s5 ) , indicative of coordination to the 
 copper(ii ) center . in the uv / vis spectrum , a single intense band ( ca . 900 dm mol cm ) 
 appears at 390 nm assignable to an azide to copper charge 
 transfer band at a mononuclear copper site . the appearance of this band is accompanied by a small increase in 
 the intensity of the d there 
 are distinct differences in the epr spectrum of cu(ii)-bacbm33 and that of cu(ii)-tagh61 . in the 
 latter , the gz and |az| values of 2.27 and 0.0162 
 cm place gh61 squarely within the type 2 classification 
 of copper site and are also indicative of a copper(ii ) ion within 
 an axial coordination geometry . this geometry was confirmed by the 
 subsequent x - ray diffraction studies of gh61 . it is currently unclear 
 why there are such differences in the coordination geometries of the met forms of both cbm33 and gh61 , but the difference is 
 likely to be of functional significance given the differences in the 
 copper active site structures of cbm33 and gh61 ( see below ) . an amino acid sequence alignment of bacbm33 with a range of selected cbm33s is shown in figure s6 . outside the active site histidines , strict conservation 
 is observed in parts of the -sandwich and in a collection of 
 tryptophan residues adjacent to the active site , where the latter 
 is probably associated with an electron transfer function . this particular 
 conservation of tryptophans is also observed in cbp21 chitin oxidases , 
 as previously noted by vaaje - kolstad et al . active 
 site conserved residues in cbm33 depicting the occlusion 
 of cu(ii ) axial coordination sites by alanine 123 and phenylalanine 
 196 . sections of the sequence alignment with two other cbm33s ( b.a 
 = bacillus amyloliquefaciens , s.m = serratia marcescens , and e.f = enterococcus 
 faecalis ) are shown surrounding 
 each of the active site residues , which are shown in bold . their associated 
 percentage identities from the full sequence alignment shown in figure s6 are indicated by the histograms below 
 the sequences according to the key . an unusual feature in cbm33 is observed in the strict conservation 
 of an alanine residue at position 123 at the end of a  strand . the methyl group of this alanine is close to the apical coordination 
 site on the copper ion ( cuc = 3.9  ) , thereby 
 providing a degree of steric congestion at this coordination site . what makes this feature notable is that the opposite apical site is 
 occupied by a highly conserved phenylalanine side chain ( position 
 196 ) , which has a near  end - on  aspect to the copper 
 ion with a closest cuc distance of 3.7  ( figure 7 ) . the combination of the alanine and phenylalanine 
 suggests that coordination to the copper in cbm33 is restricted to 
 equatorial sites only ( figure 7 ) . this is in 
 sharp contrast to gh61 where the apical sites are occupied by the 
 oxygen atoms of a tyrosine / ate and a water molecule , where the water 
 molecule could be replaced by either substrate or an oxygen molecule , 
 as suggested by li et al . whatever the 
 reasons for the differences between cbm33 and gh61 , the contrasting 
 arrangement of amino acid side chains in the copper ions secondary 
 coordination spheres , including that of the -n - methylated n - terminal 
 histidine in gh61 , is indicative of different mechanisms of action 
 between the two enzymes . region colored red is copper active site , which is surrounded by highly 
 conserved regions consisting of residues capable of forming hydrogen 
 bonds 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 . in a further indication of alternative 
 mechanisms , the conserved 
 residues on the binding face of cbm33 differ from that of gh61s . in 
 the former , the principal conservation pattern is found in amino acid 
 side chains , which can engage in hydrogen - bond interactions with a 
 polysaccharide substrate . the two regions lie on opposite 
 sides of the copper ion , thus providing an environment that likely 
 directs a specific orientation of the substrate with respect to the 
 copper and thus any reactive oxygen species generated at the active 
 site . such a directed interaction with the substrate is in accordance 
 with the observation that cbm33s react principally at the c1 site 
 of -linked polysaccharides ; it may also offer an explanation 
 as to why only even - numbered oligosaccharides are produced by cbm33 
 insofar as the up  down  up  down orientation pattern 
 of individual glycoside units within chitin and cellulose will only 
 project c  h bonds ( from c1 ) toward the enzyme binding 
 face every other glycosidic unit , as first suggested by vaaje - kolstad 
 et al . this was shown by li et al . , who identified conserved 
 tyrosine residues on the -helical l2 loop in polysaccharide 
 oxygenases and also on residues near the copper active site ( figure 8) . the residues are 
 not only in different positions on the binding face when compared 
 to cbm33s , but also probably interact with the cellulose substrate 
 in a somewhat less directional way than the potential hydrogen - bonding 
 interactions seen in cbm33 . this is commensurate with a wider range 
 of oxidation sites on cellulose that is observed with gh61 oxidative 
 action , especially type 3 pmos , and that 
 oxidation does not only occur at the exposed c1h bonds on 
 cellulose , as appears to be the case in cbm33 . it is not possible 
 to be more definitive about the conservation 
 differences between cbm33 and gh61 because we are unable to rule out 
 the possibility of post - translational modifications on cbm33 , which 
 could introduce extra functionality at the active site , for instance , 
 oxidation of phenylalanine to tyrosine and potential methylation of 
 the n - terminal histidine . indeed , we observe here that one cbm33 sequence 
 naturally has a tyrosine in place of phenylalanine 196 . nor can we 
 completely rule out a coordinative role for a highly conserved tyrosine 
 at position 197 , although note that this appears to be unlikely given 
 the large structural disruption this would entail and the absence 
 of any conformational flexibility in this region as observed across 
 several sets of deposited coordinates . additionally , an interrogation 
 of the cazy database shows that gh61s can appear as multidomain proteins 
 in which the oxygenase domain is fused to other known cellulose - binding 
 modules ( e.g. , cbm1 ) through which binding to cellulose will be significantly 
 affected . similarly , cbm33 enzymes are 
 often appended to diverse other protein modules involved in hydrolysis 
 and binding of carbohydrates , as reviewed recently by horn et al . notwithstanding these caveats , however , a structural 
 basis for the known differences in mechanisms of action of cbm33s 
 and gh61s does emerge without the need to introduce significant post - translational 
 modifications in cbm33s , and may well therefore be a reliable basis 
 for understanding differences in reactivity . we have demonstrated that a cbm33 from bacillus 
 amyloliquefaciens binds copper(ii ) with a kd  6 nm . this binding significantly 
 stabilizes the enzyme , probably reflecting the increased formation 
 of a folded state of the protein backbone . from x - band epr spectroscopy 
 and azide binding studies , we can infer that cu - bacbm33 is a mononuclear type 2 copper enzyme , but where the copper(ii ) 
 ion has a distorted axial geometry , possibly a distorted square planar 
 configuration akin to that seen in structure of cu  zn superoxide 
 dismutase . xanes spectroscopy demonstrates that the copper is susceptible 
 to reduction and is readily photoreduced by x - rays both in solution 
 and in the solid state to the copper(i ) form , an observation supported 
 by the high redox potential of cu - bacbm33 . ( very 
 recently , structures of a cbm33 from enterococcus faecalis were released on the pdb , the deposition titles of which imply a 
 similar photoreduction of copper when coordinated by the histidine 
 brace ; pdb codes : 4alr , 4als , 4alt , 4alq , 4ale , 4alc . single - crystal 
 x - ray diffraction shows that the enzyme adopts an active site structure , 
 which has broad parallels with that reported for cu - gh61s . the copper(i ) 
 histidine brace coordination geometry in cu - bacbm33 
 is t - shaped n3 , corroborated by xanes studies that show 
 a 1s to nonbonded 4p transition characteristic of low coordination 
 number copper(i ) species . differences in the secondary coordination 
 sphere of the copper ion in cbm33s and gh61s are suggestive of a different 
 mechanism of action in the two classes of enzymes , but a fuller understanding 
 of the mechanism awaits in - depth and detailed reaction studies on 
 the enzymes and their associated small molecule model complexes . the coding sequence 
 of bacillus amyloliquefaciens cbm33 
 ( ncbi reference sequence : nc_014551.1 ) from nucleotide 82 to 621 
 was codon optimized for expression in escherichia coli , synthesized with a pelb leader sequence to direct the protein to 
 the periplasm , and cloned into the pet-11a vector , by genscript . the 
 resulting pet11a - bacbm33 construct was used to transform bl21 ( de3 ) e. coli , which were grown from a single colony in 
 six 500 ml lb cultures to an a600 of 0.4 
 at 37 c shaking at 180 rpm . the temperature was then reduced 
 to 16 c , and the cells were allowed to grow further to an a600 of 0.60.8 when expression was induced 
 by the addition of iptg to a final concentration of 1 mm . the cultures 
 were left overnight before the cells were harvested by centrifugation 
 at 11  000 g for 30 min at 4 c . cells were immediately resuspended in 5 volumes of ice cold resuspension 
 buffer ( 50 mm tris ph 8.0 , 200 mm nacl , 20% w / v sucrose ) . to isolate 
 the periplasmic fraction , 40 g of hen egg white lysozyme ( sigma - aldrich ) 
 was added for every gram of cell paste and left on ice for 1 h. 60 
 l of 1 m mgso4 was then added per gram of cell paste , 
 and the suspension was incubated for a further 20 min on ice . cell 
 debris was then removed by centrifugation at 10  000 g , 4 c for 10 min , and the supernatant containing 
 the periplasmic fraction was removed into a fresh tube . this was diluted 
 4 fold with buffer a ( 50 mm tris ph 8.0 ) and passed through a 5 ml 
 hitrap q sepharose column equilibrated in the same buffer collecting 
 the 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 column 
 and was present in the flow through , which was precipitated by the 
 addition of solid ammonium sulfate to a final concentration of 3.5 
 m and left stirring overnight at 4 c . the protein was then isolated 
 by centrifugation at 38  000 g for 30 min , the 
 supernatant was discarded , and the pellet was dissolved in 10 volumes 
 of 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 to 
 a hiload 26/60 superdex 75 column equilibrated in gf buffer ( 20 mm 
 sodium acetate ph 5.0 , 250 mm nacl ) . peak fractions containing pure 
 bacbm33 were pooled and concentrated using a sartorius 10 kda cut 
 off concentrator at 4000 g . protein concentrations 
 for all subsequent experiments were determined by measuring the a280 using an extinction coefficient of 44  920 
 m cm and a molecular weight 
 of 19  821 da . the 
 redox indicator dyes 
 thionine , tetramethylphenylenediamine ( tmp ) , and 2,6-dichloro - phenolindophenol 
 ( dcpip ) were purchased from sigma . a three - electrode setup was used 
 to determine accurately the reduction potential of the redox indicators 
 and 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 placed 
 into an all - glass electrochemical cell . cyclic voltammograms ( 50 mv 
 s ) were measured for 1 mm solutions of each of 
 the redox compounds under a flow of ar . a correction factor of eshe= esce + 0.241 
 v was used . the anodic and cathodic peak potentials , as determined 
 by the electrochemical software ( epsilon basi ) , were averaged to give 
 the midpoint reduction potentials , summarized in table s2 . to judge if the dye could reduce bacbm33 , up to 10 l of approximately 0.5 mm enzyme was injected 
 into a suba - sealed cuvette containing 150 l of reduced dye 
 solution ( absorbance < 0.2 au ) and a small stirrer bar . following 
 2 min of stirring control experiments were conducted with 
 injection of buffer instead of enzyme , and it was confirmed that cbm33 
 was required to oxidize thionine or tmp in the cuvette . continuous wave x - band frozen solution 
 epr 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 300 
 spectrometer operating at 9.072 or 9.35 ghz , with a modulation amplitude 
 of 4 g and microwave power of 5 mw . simulation parameters were as follows : gx = 2.075 , gy = 2.086 , gz = 2.255 ; |ax| 
 = 39 , |ay| = 90 , |az| = 125 g. strain : axx = 210 , ayy = 210 , azz = 210 g. linewidth : 40 g ( gaussian ) , 40 g ( lorentzian ) . gz and |az| were determined accurately from the two 
 absorptions at low field where it was assumed that these absorptions 
 were separated from other aspects of the absorption envelope . accurate determination of the gx , gy , |ax| , and |ay| was not possible due to 
 the second - order nature of the perpendicular region and the significant 
 level of conformational heterogeneity , although it was noted that 
 satisfactory simulation could only be achieved with one particular 
 set of spin hamiltonian values . xanes spectroscopy data 
 were collected on a 0.6 mm solution of cu(ii)-bacbm33 at ph 5 , which 
 had been flash - frozen to 77 k ( 0.7 mm bacbm33 was prepared in 20 mm 
 sodium acetate ph 5.0 , 250 mm nacl to which cu(no3)23h2o was added to a final concentration of 
 0.6 mm ; to produce a cu(i ) sample , ascorbate was added to a final 
 concentration of 10 mm ) . data were acquired on the sample at 90 k 
 at the b18 core spectroscopy beamline at diamond light source , oxfordshire , 
 uk . at the time of the measurement , the diamond synchrotron was operating 
 at a ring energy of 3 gev in a 10 min top - up mode for a ring current 
 of 301 ma . the beamline was equipped with a si(111 ) double crystal 
 monochromator , and harmonic rejection was achieved through the use 
 of two pt - coated mirrors operating at an incidence angle of 9 mrad . the monochromator was calibrated using the first maximum in the derivative 
 in the edge region of the xas spectra of a copper foil placed between 
 the second and third ion chambers at 8979 ev . estimated flux of beam 
 at 8 kev = 5  10 ph / s . data were collected in fluorescence 
 from 8779 to 9020 ev using a nine - channel ge solid - state detector 
 at the copper k absorption edge ( 8980 ev ) in 1 ev steps . the 
 sample was contained in a 5 mm light path ptfe 400 l cell with 
 25 m thick windows made from kapton foil . the measurements 
 were made at 90 k. the incident beam intensity was measured using 
 a 30 cm ion chamber optimized using a helium  nitrogen gas mixture 
 to absorb 30% and 70% of the beam in i0 , respectively . x - ray fluorescence 
 scans were performed on crystals , which had not previously been exposed 
 to 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 three 
 drops of cryo - protectant solution to remove excess copper prior to 
 plunging in liquid nitrogen . to produce cu(i ) control samples , 20 
 mm ascorbate was included in the cryo - protectant soaking solution . fluorescence scans were performed across the copper k - edge from 8942 
 to 9017 ev with step sizes of 5 ev from 8942 to 8962 ev , 0.5 ev across 
 the edge from 8962 to 8997 ev , and 5 ev from 8997 to 9017 ev . isothermal titration 
 calorimetry was performed using a vp - itc calorimeter ( microcal ) . typically 
 protein was present in the cell between 10 and 120 m with a 
 10-fold more concentrated solution of cucl2 in the syringe . titrations were initially performed at 283 k , but the same results 
 could later be obtained at 298 k also . after an initial 2 l 
 injection , which is discarded in the data analysis , 10 l injections 
 were used during the titration with a 5 min interval between each 
 injection at ph 5.0 and 6.0 and 8 min intervals at ph 7.0 where binding 
 was slower . the buffers used during the titrations were either 20 
 mm sodium acetate ph 5.0 , 250 mm nacl , 20 mm bis - tris ph 6.0 , 250 
 mm nacl , or 20 mm bis - tris ph 7.0 , 250 mm nacl with the cucl2 solution prepared in exactly the same buffer . heats of dilution 
 were subtracted from the data , but the analysis routinely returned 
 a substoichiometric binding of cu . modification of the injection system 
 to remove cu - containing brass components improved the stoichiometries , 
 but these rarely exceeded 0.86 . this is likely due to a portion of 
 the protein having acquired cu given the very high affinity 
 of the interaction from the glassware . to give meaningful h values ( the kds are unaffected ) , the protein concentration was therefore adjusted in the software to 
 reflect the 1:1 stoichiometry of copper to protein seen in the crystal 
 structure . differential scanning 
 fluorimetry was used to determine the melting temperature of bacbm33 with and without metal ions . stability measurements 
 were performed using an agilent mx3000p qpcr machine and the fluorescent 
 dye sypro orange ( sigma - aldrich ) diluted 2000 fold from the stock 
 solution . fluorescence was measured with excitation and emission wavelengths 
 of 517 and 585 nm , respectively . all experiments were performed in 
 20 mm sodium acetate ph 5 , 250 mm nacl with protein at 25 m , 
 and a total volume of 30 l . fluorescence was monitored while 
 increasing the temperature in steps of 1 c at 30 s intervals 
 from 25 to 96 c . the fluorescence was significantly quenched 
 in the presence of copper , but a melting curve could still be obtained . 
 melting temperatures ( tm ) were calculated 
 by fitting a sigmoidal curve to the data using the mtsa program for matlab . to form the cu complex , 
 cu(no3)23h2o apo and 
 cu cocrystallization trials were then set up in 
 parallel with a protein concentration of 7 mg / ml using a mosquito 
 robot ( ttp labtech ) . the best crystals in both cases were obtained 
 in condition d1 of the pact screen ( qiagen ) : 0.1 m mmt buffer ph 4.0 , 
 25% peg-1500 . crystals used to determine the cu - bound / ascorbate 
 soaked structure were cocrystallized with copper in the same way as 
 described above . crystals were cryo - cooled for 
 data collection by first soaking 
 for 30 s in mother liquor supplemented with 20% v / v ethylene glycol 
 before plunging directly in liquid nitrogen . for the ascorbate soak , 
 20 mm sodium ascorbate was included in the cryo - protectant . diffraction 
 data were collected at diamond light source , beamlines i04 - 1 and i03 
 at wavelengths of 0.917 and 0.976  , respectively . data were 
 indexed and integrated using xds with 
 subsequent processing performed using the ccp4 software package . the serratia marcescens cbp21 structure ( pdb 2ben ) was prepared as a molecular 
 replacement search model using chainsaw , cutting back side chains to their nearest common atom . the apo and cu - bound structures were initially determined by 
 molecular replacement using phaser , both 
 containing two molecules in the asymmetric unit . following structure 
 solution , pseudotranslational symmetry was detected in the apo - bacbm33 data . the structure was therefore solved again 
 using molrep inputting the pseudo translation vector 0.500 , 0.000 , 
 0.127 for the off - origin patterson peak . arpwarp was then used to rebuild the initial models before subsequent 
 manual building and refinement using coot and refmac5 , respectively . the ascorbate soaked crystals were isomorphous to the copper - free 
 crystals , and so the copper - free structure was simply refined against 
 these data , with waters and flexible loops removed , to yield the structure . all data processing and refinement statistics can be found in table s1 . coordinates and accompanying structure 
 factors for the apo- , cu - bound , and cu - bound / ascorbate 
 soaked enzyme have been deposited in the protein data bank with accession 
 codes 2yow , 2yox , and 2yoy , respectively .
the 
 capacity of metal - dependent fungal and bacterial polysaccharide 
 oxygenases , termed gh61 and cbm33 , respectively , to potentiate the 
 enzymatic degradation of cellulose opens new possibilities for the 
 conversion of recalcitrant biomass to biofuels . 
 gh61s have already 
 been shown to be unique metalloenzymes containing an active site with 
 a mononuclear copper ion coordinated by two histidines , one of which 
 is an unusual -n - methylated n - terminal histidine . 
 we now report 
 the structural and spectroscopic characterization of the corresponding 
 copper cbm33 enzymes . 
 cbm33 binds copper with high affinity at a mononuclear 
 site , significantly stabilizing the enzyme . 
 x - band epr spectroscopy 
 of cu(ii)-cbm33 shows a mononuclear type 2 copper site with the copper 
 ion in a distorted axial coordination sphere , into which azide will 
 coordinate as evidenced by the concomitant formation of a new absorption 
 band in the uv / vis spectrum at 390 nm . 
 the enzyme s three - dimensional 
 structure contains copper , which has been photoreduced to cu(i ) by 
 the incident x - rays , confirmed by x - ray absorption / fluorescence studies 
 of both aqueous solution and intact crystals of cu - cbm33 . 
 the single 
 copper(i ) ion is ligated in a t - shaped configuration by three nitrogen 
 atoms from two histidine side chains and the amino terminus , similar 
 to the endogenous copper coordination geometry found in fungal gh61 .