density functional study on uv/vis spectra of copper-protein active sites: the effect of mutations

Density Functional Study on UV/VIS Spectra of Copper-Protein Active Sites:The Effect of Mutations

by Marcel Swart*a)b) and Mikael P. Johanssona)

a) Institut de Qu�mica Computacional & Departament de Qu�mica, University of Girona,Campus Montilivi, ES-17071 Girona

b) Institucio Catalana de Recerca i Estudis AvanÅats (ICREA), Pg. Llu�s Companys 23,ES-08010 Barcelona (e-mail: [email protected])

UV/VIS Electron excitation spectra have been computed for large, realistic model systems of theblue copper protein family. Fully quantum-chemical calculations at the density-functional theory levelemploying polarized triple-z basis sets have been performed on systems of over 120 atoms, withoutsymmetry. Different mutants, with the ligating methionine of the wild type Cu center exchanged forhistidine (M121 H) and glutamine (M121Q), have been investigated in order to obtain insight about howthe influence of the exact surrounding milieu of the Cu-atom affects the computed spectrum. Withsufficiently large model sizes, inclusion of the environment by using continuum solvation models do notchange the spectra significantly. More direct and rigorous treatments are needed to reliably assess theeffect of the surrounding protein on the electronic structure of the active sites.

Introduction. – Members of the family of blue copper proteins (BCPs), also knownas type-1 Cu proteins, are, as the name suggests, characterized by an intense blue color[1]. This spectroscopic feature is produced by a specific active-site arrangement ofamino acids surrounding the Cu ion: there are two histidine and one cysteine ligandssituated more or less in a plane, with one methionine axial ligand above the plane and,in some cases, a fifth axial glycine ligand below the plane. This active-site arrangementis a compromise between the preferred coordinations of CuI and CuII, which enablethese BCPs to efficiently switch between these two Cu oxidation states, and hence theirfunction of electron-transfer (ET) proteins.

One of the most studied members of the BCP family is azurin, a relatively smallprotein containing 128 or 129 residues (depending on the organism from which it isobtained). It has mainly b-sheet regions, but also contains an a-helix, and has the activesite at the top end (see Fig. 1).

The Cu ion in Pseudomonas aeruginosa azurin is surrounded in the plane by His46,Cys112, and His117, and has two axial Gly45 and Met121 ligands (see Fig. 2). Becausethe azurin protein is a very stable protein, even after single-point mutagenesis of one ofthe Cu ligands, it has been used extensively in the past decades. Single and doublemutants have been prepared [2– 5] (of either residues in or close to the active site, or faraway from them), chemical linkers have been used to bind two azurin moleculestogether [4 –7], transmetallation by Cd, Co, Zn, and Mn have been performed [8 – 16],and many crystal structures are available for, e.g., reduced (CuI), oxidized (CuII), andthe apo-protein. Canters and co-workers [17– 19] prepared single mutants (His117Gly)

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)1728

� 2012 Verlag Helvetica Chimica Acta AG, Z�rich

to establish the role of the surface-exposed His117 residue, which exhibits a green color.Upon treatment with exogenous ligands (chloride, bromide, imidazole), the character-istic blue color of the BCP family is restored. As azurin has only one tryptophanresidue, it has also been used in fluorescence studies [20 – 22].

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012) 1729

Fig. 1. Azurin protein (from Pseudomonas aeruginosa) with active site indicated in sticks, and copper ionas orange ball

The axial ligands (Met121, Gly45) were shown to exert a large influence on thespectroscopic properties, with different members of the BCP family showing a largevariety in reduction potentials. The nature of the interaction between the axial ligandsand the Cu ion, and indeed if the Gly45 backbone carbonyl should be regarded asligand, were unknown. Early crystal structures placed the Cu�S (Met121) distance inazurin at ca. 3.1 �, as in the 5AZU PDB structure of 1.9 � resolution [23], while a morerecent and slightly higher resolution one, 1DYZ at 1.75 �, suggests a distance closer to3.3 � [24]. This latter distance is very different from the one observed in, e.g.,plastocyanin, another BCP protein that does not have an glycine backbone carbonyl asfifth ligand. An early study by van Gastel and co-workers [25] showed that replacementof the Met121 ligand, by either glutamine or histidine, have a large impact on the UV/VIS spectra. This was further explored by Honek, Lu, and co-workers [26] who usedunnatural amino acids at the 121 position, and studied their effect on the reductionpotential and the UV/VIS spectra.

The effect of protein strain on the properties of the azurin family have beenextensively studied computationally by Ryde et al., and Rothlisberger and co-workers[27 – 29]. While the protein environment was found to have little effect on the geometry[27] [28], the redox potential was shown to be greatly affected by the proteinenvironment [29].

The weakly interacting Gly45 was supported by Richards, Gray, and co-workers[30], who used double mutants at the 112 and 121 positions. In the C112D/M121L case,the Cu�O (Gly45) distance is reduced to 2.35 �, and hence Gly45 should be regardedas a true ligand to the metal. The resulting protein was dubbed to be of �type 0�, since itsspectroscopic characteristics do not resemble either one of the other types. In the sameweek, another study was reported by Lu and co-workers [31], who showed that twosecondary-coordination-sphere interactions (involving Asn47 and Phe114) were vitalfor understanding the reduction potential of these cupredoxin proteins. The N47Smutation affects the rigidity of the Cu-binding site and, probably, the direct H-bonds

Fig. 2. Active site of Pseudomonas aeruginosa azurin protein


between the protein backbone and Cys112. Introducing a H-bond donor at position 114(F114N) perturbs H-bonding near the Cu site, while deleting a direct H-bond to Cys112(F114P) results in a lower redox potential. Mutations at these two positions, togetherwith a third mutation at the 121 position, then led to a cupredoxin with the highestreported reduction potential of þ706 mV vs. a normal hydrogen electrode (NHE)[31].

One of us (M. S.) previously studied [32] the UV/VIS spectra of wildtype azurinand two mutants (M121 H, M121Q) within hybrid quantum-mechanical/molecular-mechanical (QM/MM) calculations using a polarizable force field (DRF approach[33]) for the MM part, and the semi-empirical ZINDO (Zerner�s intermediate neglectof differential overlap) method [34] for the QM part. At that time, the time-dependentdensity-functional theory (TD-DFT) method for computing electronic excitations wasnot available for use on spin-unrestricted systems, e.g., the CuII active site. The resultswith ZINDO indicated that the presence of the protein environment was vital forobtaining UV/VIS spectra that correspond well with experimentally observed ones.Since then, unrestricted TD-DFT has become available, and, recently, Swart et al.presented a new density functional (SSB-D) [35], which, among other things, performsexceptionally well for relative spin-state energies [36] [37]. Here, we investigate theUV/VIS spectra of active-site models of azurin and two mutants using large basis setsand the SSB-D functional. These calculations are among the largest and most resource-consuming quantum-chemical excitation energy calculations performed to date.

Computational Details. – Density-functional theory (DFT) calculations were performed with Tur-

bomole [38–42] version 6.2 (for the geometry optimizations) and the Amsterdam Density Functionalprogram ADF [43] [44] version 2010.01 (for TD-DFT calculations). Energies and gradients during thegeometry optimizations were computed at the generalized gradient approximation (GGA) level [45],using the Perdew�Burke�Ernzerhof functional [46], including Grimme�s dispersion correction [47](PBE-D). For geometries, we used the def2-SVP basis on all atoms, except Cu for which we used thedef2-TZVP basis [48] [49]. Subsequently, the time-dependent DFT (TD-DFT) calculations wereperformed with the SSB-D functional [35] using an uncontracted set of Slater-type orbitals (STOs) oftriple-z quality and one set of polarization functions (TZP) [50].

Results and Discussion. – We have optimized the geometry of the active-site modelsof wildtype azurin and its mutants, while keeping a number of C-atoms of the amino-acid residues kept frozen at the positions they have in the protein (PDB code 5AZU[23]; chain D). By keeping these peripheral atoms frozen, we simulated the strainimposed on the active site residues by the protein environment. In addition to the C-atoms, it also turned out that the peptide N-atom connecting Thr113 and Phe114 had tobe frozen; a free optimization cleaved the N�H· · ·S H-bond to Cys112, leading tostructures very different from those found in the crystal structures. A more realisticsimulation, which would obliviate the need for the structural constraints, would be toinclude the protein environment explicitly in hybrid QM/MM calculations.

The model systems include all Cu ligands (Gly45, His46, Cys112, His117, andMet121) and two secondary coordination sphere residues (Asn47 and Phe114) that areH-bonded to the S-atom of Cys112. In total, the system contains 121 atoms. For the twomutant active site models (M121 H and M121Q), we started from the same initialgeometry and kept the same atoms frozen. Therefore, any differences with respect to


the wildtype active site resulted directly from changing the axial residue at position 121(note that none of the atoms of this latter axial residue have been frozen).

The geometries of the three model systems are very similar (see Fig. 3), which is notthat surprising given that the same initial geometry and frozen atoms were used (videsupra). Nevertheless, the difference in orientation of the axial ligand at position 121does have an effect on the position of the Cu ion, both of which have an effect on theCu�ligand distances (see the Table). The main effect of the difference in the axialligand is found for the Cu�L121 distance, as was expected. For the wildtype active sitemodel, this distance is found at 2.64 �, i.e., much smaller than the distance within theprotein (3.13 �) and also much smaller than was obtained in hybrid QM/MMcalculations previously by one of us (3.18 �). It should be noted that here we includedispersion corrections, which were not included in the previous QM/MM calculations;how important these corrections are will become clear in our future QM/MM studies.Moreover, the value obtained here is more similar to the value reported forplastocyanin, a BCP protein that lacks the axial Gly45 ligand. The active-site modelsof the two mutants show remarkably different Cu�L121 distances, 2.03 (M121 H) and2.23 � (M121Q), respectively. This shortening of the axial ligand bond is also observedin the crystal structures of these mutants, where they have values of 2.22 (M121 H) [51]and 2.26 � (M121Q) [52], respectively.

The axial fifth ligand, Gly45, is found also at different distances from Cu for thethree systems; for the wildtype active-site model, it is at 3.24 � from Cu (see the Table),while, for the M121 H mutant, it is at 4.02 �. This drastic increase is also observed in theM121 H crystal structure, and, hence it reflects a remarkable similarity between themodel systems used here, and the actual active site as they are found inside the proteinstructures.

The different axial ligands will naturally have an effect on the electron distributionin the system. This is exemplified in the spin-density distribution of the ground state,


Table. Structural Parameters [�] in the Three CuII Active-Site Models and Azurin Proteins

Starting structure Wildtype M121 H M121Q

Proteina) Model Proteinb) Model Proteinc) Model

d(Cu�ligand)Cu�O(G45) 3.020 2.925 3.235 3.886 4.015 3.372 3.213Cu�N(H46) 2.074 2.062 1.989 2.017 2.056 1.936 1.989Cu�S(C112) 2.255 2.258 2.186 2.163 2.286 2.120 2.188Cu�N(H117) 1.986 2.031 1.998 2.080 1.997 2.046 1.996Cu�L(X121) 3.076 3.131 2.644 2.220 2.026 2.263 2.227

H-Bonds SC112d)

N47 3.582 3.541 3.681 3.597 3.475 3.419 3.635F114 3.466 3.515 3.530 3.688 3.610 3.672 3.475

a) PDB Code: 5AZU [23], average over active sites of four chains. b) PDB Code: 1A4A [51], averageover active sites of two chains. c) PDB Code: 1URI [52], average over active sites of two chains.d) Indicated are SC112�Nbackbone-47/114 distances.

depicted for the three systems in Fig. 4. For the wildtype enzyme, the fourth ligand,methionine, is almost closed-shell, with very little spin density. The N-atom of thehistidine of the M121 H mutant, on the other hand, receives the pacifier-shaped spindensity typical to histidine residues connected to an open-shell transition metal. Theglutamine of M121Q is again almost closed-shell. For all of the systems, the S-atom ofthe cysteine residue connected to Cu exhibits a significant unpaired spin population inits p-shell. With these obvious differences in the ground state, the electronic excitationsare expected to be quite different, affecting the UV/VIS spectra discussed below.

UV/VIS Spectra. With the structures of the active-site models as described above,we have then calculated the UV/VIS spectra for the three systems to see if we can

Fig. 4. Unpaired spin density in the models of wildtype azurin (5AZU) , and the M121 H and M121Qmutants


Fig. 3. Superposition of active-site models of wildtype azurin (colored by element), and the M121 H (ingreen) and M121Q (in blue) mutants

reproduce the changes, induced by the mutations, on these spectra. Experimentally, adramatic shift was observed both for the shape and the peak positions of the UV/VISspectra for these proteins. The wildtype protein has an intense single peak at 625 nm,which changes to two peaks at 440 and 590 nm (with intensity ratio of 2.1 :1.0) for theM121 H mutant, and to two peaks at 450 and 610 nm (intensity ratio of 0.13 :1.0) for theM121Q mutant. We are interested in trying to separate two issues: i) the intrinsic effectof the mutation of one of the active-site ligands on the UV/VIS spectra, and ii) theeffect of the protein environment on these spectra. The second point will be clarified inmore detail in the near future, when we will also include the protein environmentexplicitly in hybrid QM/MM calculations. A quick (but maybe not so accurate) probeof the effect of the environment might be obtained by including a dielectric continuummodel (COSMO [53], dielectric constant e of 4.0, solvent radius of 1.3 �) within theTD-DFT calculations. As depicted in Fig. 5, including a dielectric continuum does nothave a large effect on the obtained spectrum. It should, however, be mentioned thatthere is a discussion on what would be an appropriate value for the dielectric constantof a protein environment. A �standard� e of 4.0 value is often invoked [54] [55], whilefor some systems, molecular-dynamics simulations suggest a value of e in the range of25– 30 [56] [57]. For this reason, Siegbahn and Himo propose to increase the size of theQM system, until the actual value of the dielectric constant does not matter any longer[58]. Nevertheless, as seen from Fig. 5, the effect of including the dielectric continuumis very small, suggesting that the model-system sizes here are reasonably large already.

When now comparing the experimental [25] (see Fig. 6) and computed (see Fig. 7)UV/VIS spectra for the three proteins, a number of features stand out. First, there is notonly one peak observed for the active-site model of wildtype azurin, but at least four;moreover, the most intense peak is blue-shifted to ca. 540 nm. Second, the most intensepeak of the M121 H active-site model is found at a position by ca. 120 nm lower than themaximum peak of the wildtype model. Third, the most intense peak of the M121Q

Fig. 5. UV/VIS Spectra obtained from TD-DFT for active-site model of wildtype azurin (5AZU) in thegas phase and embedded in solvent continuum model (COSMO) with dielectric constant e of 4


model is found at almost the same position as the wildtype model. Therefore, theexperimental trends may be considered to be reproduced reasonably (given theabsence of the protein environment in our calculations), apart from the finding that weobserve many more peaks than one should expect from the experimental studies [25].However, it is also well-documented that standard pure density functionals (to whichclass the SSB-D functional belongs) in general underestimate the energy level ofexcited states [59]. Therefore, instead of inspecting the blue-shifted region, we shouldbe focusing our attention to the red-shifted region at longer wavelengths. Indeed, if wezoom in the region between 650 and 750 nm (see Fig. 8), we observe a similar pattern:


Fig. 6. Experimental UV/VIS spectra for wildtype, M121 H, and M121Q azurin. Adapted from [25] (withpermission of the American Chemical Society).

Fig. 7. UV/VIS Spectra obtained from TD-DFT for active-site models of wildtype azurin (5AZU) and twomutants (M121 H and M121Q)

the major peak of the M121Q model corresponds well with one of the peaks of thewildtype model, while the major peak of the M121 H model is found at lowerwavelengths.

Nevertheless, there are some oddities, such as the finding that the wildtype modelshows two peaks instead of one, and reversely, that the M121Q model shows only oneinstead of two. Also, the difference in wavelength between the M121 H mutant model,on the one hand, and the other two protein models, on the other, amounts to some 70wavenumbers instead of the experimentally observed 180 wavenumbers. Thesedifferences between computed and experimental spectra might very well be resultingdirectly from the absence of the protein environment in our calculations. Indeed, one ofus showed previously that both the shape and peak positions differ dramatically in thepresence or absence of the protein environment [32]. Another possible source of thedifference is that, with the current restraints, the geometry of the immediate Cu-sitemight be suboptimal. Solomon, Pierloot, and co-workers have found that, while theenergy difference between more tetragonal and more trigonal structures can be verysmall, the computed excitation spectra can, nevertheless, differ significantly [60 – 63]We are currently investigating this further through the use of QM/MM calculations toinclude the protein environment as well, thus obliviating the need for any artificialgeometry constraints.

Conclusions. – UV/VIS Electron-excitation spectra have been computed for large,realistic model systems of the blue copper protein family. Fully quantum-chemicalcalculations have been performed on systems including over 120 atoms, withoutsymmetry. Different model systems have been investigated to obtain insight about howthe influence of the exact surrounding milieu around the Cu-atom affects the computedspectrum. With model systems of this size, the indirect inclusion of environmentaleffects by means of a continuum model do not affect the spectra significantly. More


Fig. 8. Zoom of most relevant part of the UV/VIS spectra for wildtype (5AZU) , M121 H, and M121Qazurin as obtained from TD-DFT calculations at SSB-D/TZP/COSMO level

elaborate means of including the protein matrix in the calculations could, however,have a notable effect on the electronic properties of the active sites. When computingelectronic excitation energies for large systems with open electronic shells and unpairedelectron spins, it is a priori difficult to estimate how many excitations need to beobtained. To span the relevant energy region of excitations down to wavelengths of250 nm, 100– 150 individual excitations have to be computed, with the system sizes andtheoretical methods presented here.

The following organizations are acknowledged for financial support: the Ministerio de Ciencia eInnovacion (MICINN, Project Nos. CTQ2008-06532/BQU and CTQ2011-25086/BQU), the DIUE of theGeneralitat de Catalunya (project No. 2009SGR528), and the European Fund for Regional Development(FEDER, grant UNGI08-4E-003). M. P. J. was further supported by a MICINN Juan de la CiervaFellowship (project JCI-2009-05953) and The Academy of Finland (project 136079). The authors aregrateful to the computer resources, technical expertise, and assistance provided by BSC, the BarcelonaSupercomputing Center – Centro Nacional de Supercomputacion.

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Received February 13, 2012


density functional study on uv/vis spectra of copper-protein active sites: the effect of mutations

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