Protein–Support Interactions for Rationally ... - ACS Publications

Article pubs.acs.org/JPCB

Protein−Support Interactions for Rationally Designed Bilirubin Oxidase Based Cathode: A Computational Study Ivana Matanovic,†,‡ Sofia Babanova,†,§ Madelaine Seow Chavez,† and Plamen Atanassov*,† †

The Department of Chemical and Biological Engineering, Center for Micro-Engineered Materials (CMEM), University of New Mexico, Albuquerque, New Mexico 87131, United States ‡ Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, United States § J. Craig Venter Institute, La Jolla, California 92037, United States S Supporting Information *

ABSTRACT: An example of biocathode based on bilirubin oxidase (BOx) was used to demonstrate how density functional theory can be combined with docking simulations in order to study the interface interactions between the enzyme and specifically designed electrode surface. The electrode surface was modified through the adsorption of bilirubin, the natural substrate for BOx, and the prepared electrode was electrochemically characterized using potentiostatic measurements. The experimentally determined current densities showed that the presence of bilirubin led to significant improvement of the cathode operation. On the basis of the computationally calculated binding energies of bilirubin to the graphene support and BOx and the analysis of the positioning of bilirubin relative to the support and T1 Cu atom of the enzyme, we hypothesize that the bilirubin serves as a geometric and electronic extension of the support. The computational results further confirm that the modification of the electrode surface with bilirubin provides an optimal orientation of BOx toward the support but also show that bilirubin facilitates the interfacial electron transfer by decreasing the distance between the electrode surface and the T1 Cu atom.

1. INTRODUCTION Multicopper oxidases (MCOs) have been extensively exploited as electrocatalysts for oxygen reduction in a variety of bioelectrochemical systems due to the fact that they offer several advantages over commonly used inorganic catalysts such as platinum.1−3 Enzymes from this family are highly specific and can effectively couple the oxidation of a wide range of substrates with the reduction of molecular oxygen. In addition, they have high redox potential for oxygen reduction and can operate at biologically benign conditions. Bilirubin oxidase (BOx) from Myrothecium verrucaria, for instance, efficiently catalyzes the conversion of bilirubin to biliverdin with the subsequent reduction of molecular oxygen to water. The active sites of MCOs share common features. Usually they are characterized by four redox active Cu atoms, which are ascribed as type 1 (T1), type 2 (T2), and type 3 (T3) based on their spectroscopic signatures. The Cu atoms are arranged in a similar manner with one T1 Cu center that is located near the protein surface and approximately 13 Å from remaining three Cu atoms, which form a trinuclear copper cluster (TNC) active site. The molecular structure, thermodynamic and spectroscopic properties, and the role Cu centers play in the enzyme’s activity have been extensively studied.4−7 It is known that the T1 site is involved in electron transfer from the substrate, while the TNC is the site where the molecular oxygen binds and is reduced to water.6,7 Immobilization and optimal interaction of a biocatalyst with the support material is one of the key components in the design © 2016 American Chemical Society

of enzyme based electrodes for efficient bioelectrochemical applications.2,8−14 It is known that the efficient 4e- mechanism of oxygen reduction reaction (ORR) to water by MCOs requires the enzyme to be oriented with its T1 center situated in the proximity of the electrode surface.15−17 The direct electron reduction of oxygen by MCOs starts with an interfacial electron transfer from the electrode to the T1 center. Electrons are then shuttled by a Cys-2His pathway to the TNC site where oxygen is reduced to water.6 Two channels from the surface of the enzyme to the TNC center enable the exchange of oxygen and water between the TNC center and the surrounding solvent. It has been determined that for MCOs the rate of oxygen turnover is determined mainly by the interfacial electron transfer between the support and the T1 center.18 Based on this knowledge, several approaches for the development of efficient biocathodes based on MCOs were previously proposed. Blanford et al. proposed to use derivatives of anthracene while Giroud et al. proposed to use anthracenemodified pyrenes to immobilize and orient T. versicolor laccase on mesoporous carbon supports.19,20 These two approaches use the same principle in which the modifier binds to the hydrophobic pocket of the enzyme close to the T1 center. As there are several other hydrophobic pockets on the surface of these enzymes, which are not close to T1 center, these Received: February 16, 2016 Revised: March 23, 2016 Published: March 25, 2016 3634

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

enzyme, its substrate, and the electrode. Namely, due to the bilirubin’s specific 3D structure, interaction of the enzyme with bilirubin adsorbed on a carbonaceous support induces large rearrangements in the enzyme’s binding pocket and alters the way bilirubin interacts with the surface of the electrode. It was also concluded that in the case of bilirubin-modified BOx electrode bilirubin serves as a geometric and electronic extension of the electrode. Because of the beneficial positioning of bilirubin toward both the enzyme and the support material, modification of the electrode surface with bilirubin facilitates the electron transfer from the support to the Cu atom of the T1 center and thus enhances the overall cathode performance in terms of generated current densities.

modification procedures can also lead to the orientations of MCOs with the T1 center away from the electrode, which are not as efficient. Our group, on the other hand, proposed to use surface modification procedure based on the “key lock principle” that relies on the specific interactions between the enzyme and its natural substrate. For example, syringaldazine and bilirubin were used to modify the support of the cathodes based on laccase or bilirubin oxidase demonstrating approximately 3−9 times increase in current density, respectively.10,21 In addition, it was shown that the simultaneous tethering of the enzyme to the electrode surface using tethering agents can provide stability, improve reproducibility, and to some extent provide proper orientation of the enzyme.11 Although significant advances were made in the design of MCO based cathodes over the past decade, additional improvements are needed to create bioelectrodes with higher activity, stability, and ease of production that will lead to their broader application. Rational design of electrode surface modifications provides one of the most promising routes for creating MCO based cathodes with enhanced performances. Such a design, however, demands deeper understanding of the efficient enzyme positioning on the electrode surface and the role that the modifier molecule plays in the interfacial electron transfer. Computational approaches could play a key role here by allowing for an understanding of the system at the molecular level.12,22 Bilirubin oxidase from M. verrucaria represents an ideal model system for such a study. Namely, the complete crystal structure of the enzyme is known,4,5 and the structural details of the substrate binding site were also described.5 The work by Cracknell et al. also showed that electrode modified with bilirubin, a natural substrate of BOx, has more than a double electrochemical response as compared to the electrode with the enzyme adsorbed to unmodified graphitic surface.5 In addition, it was reported by our group that the electrode modification with 1-pyrenebutanoic acid succinimidyl ester and 2,5-dimethyl-1-phenyl-1H-pyrrole-3-carbaldehyde, a functional analogue of bilirubin, induces a 20 times increase in the generated current density compared to the unmodified BOx cathode.10 Both modifying procedures exploit the fact that the substrate’s binding pocket is close to the T1 Cu center of BOx; however, the mechanism of the observed enhancement remains unknown. It is not clear whether the bilirubin or its analogues promote adsorption of BOx on the electrode surface in the orientation that enhances direct electron transfer from the electrode to the enzyme or whether these molecules participate in the interfacial electron transfer by serving as electron mediators. We previously used molecular docking simulations to determine the role of 1,2-benzoquinone, 1,4-benzoquinone, and ubiquione on the operation and mechanism of electron transfer in PQQ-dependent soluble glucose dehydrogenase anodes.22 This was the first time docking simulations were used to gain fundamental understanding of the interactions between the support, modifier molecule, and the enzyme. In addition, the role of the modifier molecules in the design and operation of the enzymatic bioanodes was explained. In this work we use a similar approach to study the bionano interface in BOx based biocathode and to determine the role of bilirubin in the observed enhanced electrocatalytic reduction of oxygen. Furthermore, for the first time a comparison between the docking and density functional theory results was made in order to validate the predictions of the docking simulations. Our results reveal highly dynamic interaction between the

2. EXPERIMENTAL SECTION 2.1. Cathodes Preparation. MWBP-PBSE-BOx cathode: Multiwall buckypaper was cut (d = 0.3 mm) and immersed in 10 mM solution of 1-pyrenebutanoic acid succinimidyl ester (PBSE) in DMSO and left for 1 h. After the PBSE immobilization, the electrode was carefully washed to take out the unattached PBSE and immersed in 10 mg/mL solution of bilirubin oxidase and kept for 16 h at 4 °C. The final electrode was washed and tested in 0.1 M phosphate buffer saturated with oxygen. MWBP-PBSE-bilirubin-BOx cathode: Multiwall buckypaper was cut (d = 0.3 mm) and immersed in 10 mM PBSE solution in DMSO and left for 1 h. After the PBSE immobilization, the electrode was washed with DI water and immersed in 10 mM bilirubin aqueous solution for another hour. The electrode was carefully washed to take out the unattached bilirubin and immersed in 10 mg/mL solution of bilirubin oxidase where it was kept for 16 h at 4 °C. The final electrode was washed and tested in 0.1 M phosphate buffer saturated with oxygen. The two electrodes differed regarding the presence or absence of bilirubin. 2.2. Electrochemical Characterization. A three-electrode setup was used for the electrochemical characterization of the BOx based cathodes, which was used as working electrodes with Ag/AgCl and Pt wire as reference and counter electrodes. The open circuit potential (OCP) of the electrodes was measured for 1 h before the electrode polarization. Potentiostatic polarization curves were acquired from OCP to 0 mV with a step of 50 mV. Each potential was applied for 300 s, and the steady state-current was recorded. The electrochemical experiments were performed under room temperature, and the electrolyte was constantly purged with pure O2. The counter electrode had a geometric surface area (GSA) of 0.19 cm2 while the working electrode had GSA of 0.28 cm2, which is comparable to the surface area of the counter electrode. 2.3. Calculation Details. Optimized structures of bilirubin and biliverdin in solution were obtained using B3LYP/6311+G(d,p) level of theory with polarizable continuum model as implemented in Gaussian 09 quantum chemical package.23 Geometries of bilirubin and biliverdin adsorbed on a carbonaceous support were optimized using gradient approximation (GGA) to density functional theory (DFT) with the vdW-DF functional proposed by Dion et al.24,25 and projector augmented-wave pseudopotentials25−27 as implemented in Vienna Ab initio Software Package (VASP).28−31 A carbonaceous support was modeled as a graphene sheet with a (6 × 6) supercell with dimensions of 25.56 Å × 25.56 Å and a vacuum region of 20 Å. Electronic energy was calculated using 3 × 3 × 1 k-point Monkhorst−Pack mesh32 and the tetrahedron 3635

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

3. RESULTS AND DISCUSSION 3.1. Electrochemical Measurements. BOx based oxygen reducing cathode was first electrochemically characterized in the presence and absence of bilirubin. The OCP of the MWBPPBSE-BOx cathode was determined to be 539 ± 3 mV vs Ag/ AgCl, and the OCP for the cathode modified with bilirubin was 554 ± 2 mV vs Ag/AgCl. The higher OCP of the MWBPPBSE-bilirubin-BOx cathode can be used as an indication for improved contact of the electrode surface with the T1 center of the enzyme. Along with the increased OCP of the cathode in the presence of bilirubin, the activity of the cathode was also enhanced (Figure 1). The generated current densities, when

method with Blöchl corrections. Plane-wave basis cutoff was set to 400 eV. The charge transfer was calculated by analyzing the electronic charges, which were obtained using Bader’s analysis of charge densities33−35 with 9 × 9 × 1 k-point Monkhorst− Pack mesh. The adsorption energy was calculated as the electronic energy of the graphene surface with bilirubin adsorbed minus the electronic energy of an unmodified surface minus the electronic energy of an isolated molecule. Docking simulations of bilirubin to BOx from M. verrucaria were performed using molecular docking software AutoDock Vina. 36,37 AutoDock Vina is an improved version of AutoDock4.237 with a new knowledge based statistical scoring function. The advantages of AutoDock Vina include improved prediction accuracy and speed, which is not only due to the simplified scoring function but also due to its multicore capability. A crystal structure of bilirubin oxidase from RCSB Protein Data Bank (2XLL) was used as the 3D structure of the enzyme. The search space was set as 32 Å × 32 Å × 32 Å and was situated around the experimentally determined binding pocket of bilirubin, which included T1 Cu atom. In all cases, docking simulations were performed at least a half dozen times with increasing exhaustiveness. Obtained binding models were analyzed using AutoDockTools.38 The most frequent model with the lowest binding energy was determined as the docking site of a certain ligand to BOx. Docking simulations were performed with both rigid and flexible bilirubin. In addition, the enzyme was also set either as rigid or flexible by allowing for selective receptor flexibility. The flexibility was achieved by allowing rotations around torsional degrees of freedom in the ligand and/or in the side chains of the amino acids in bilirubin oxidase. However, note that the binding affinities obtained from the rigid and flexible substrate dockings cannot directly be compared. A binding affinity of a flexible ligand is always smaller than the binding affinity of the equivalent rigid ligand in the same bound conformation. In AutoDock Vina flexibility is penalized because it allows the scoring function to fit the training data set better as it accounts for certain entropic effects. The density functional theory calculations of the interaction between the BOx pocket and bilirubin were performed using the B3LYP/6-31G level of theory with Gaussian 09 quantum chemical package. The model of the binding pocket of bilirubin to BOx included all the experimentally determined amino acids in the binding pocket, T1 Cu atom, and all the amino acids coordinated to the Cu atom (ASN197, SER198, TRP200, SER231, SER233, MET273, GLY304, THR305, ASP306, TRP361, GLY395, TRP396, THR396, THR397, HIS398, PRO399, TRP433, ASN459, HIS462, MET467, and CYS457). The input geometry was obtained by taking a crystal structure of BOx from RCSB Protein Data Bank (2XLL) and by removing all the amino acids that are not identified as a part of the binding pocket. Dangling bonds were replaced by hydrogen atoms, and bilirubin was placed into the BOx pocket using Visual Molecular Dynamic program.39 The structure was then allowed to relax. In order to prevent the structure to collapse, carbon atoms of dangling C-termini and N atoms of dangling N-termini were held fixed. The adsorption energy was calculated as the difference in the electronic energy between the BOx pocket optimized with bilirubin and the sum of the electronic energy of the BOx pocket without bilirubin and the electronic energy of an isolated bilirubin molecule.

Figure 1. Potentiostatic polarization curves measured with BOx cathode with (red) and without (black) support modification with bilirubin. The error bars represent the standard deviation calculated from the electrochemical measurement of three replicate electrodes.

the enzyme was properly oriented, increased from 0.56 ± 0.23 mA/cm2 without bilirubin to 1.25 ± 0.17 mA/cm2 when the electrode surface was modified with bilirubin. We can, thus, conclude that the presence of bilirubin as an orienting agent led to significant improvement of the cathode operation (P = 0.01). 3.2. Interaction between Bilirubin and the Electrode Surface. In order to understand the mechanism of the observed enhancement in the current density of the BOx biocathode modified with bilirubin, we fist used DFT to study the interaction between bilirubin and the support material, which was modeled as an infinite graphene sheet. Figure 2 shows DFT optimized structures of bilirubin in water and

Figure 2. DFT optimized structures of bilirubin in water and adsorbed on the graphene surface. 3636

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

Figure 3. Left: total density of states of pristine graphene (black) and graphene with bilirubin adsorbed (red). Right: total density of states of pristine graphene (black) and graphene with biliverdin adsorbed (red).

bilirubin adsorbed on the graphene surface. In solution, the bilirubin molecule has a butterfly like shape with two molecular planes joined by the central methylene bridge (Figure 2a). Each molecular plane is characterized by three intramolecular hydrogen bonds. A bifurcated hydrogen bond exists between the carboxylic group and two imino groups, and a linear hydrogen bond forms between the carboxylic group and the carbonyl group.40 In the DFT optimized structure, N−H···O, N−H···O, and O−H···O distances are determined as 2.68, 2.78, and 2.53 Å, respectively. The existence of these hydrogen bonds is confirmed in previous spectroscopic and chemical studies of bilirubin.41 Optimized geometries further show that bilirubin does not change its shape significantly when interacting with the graphene surface (Figure 2b). The molecule orients itself with one of the molecular planes almost parallel to the surface with the closest distance between bilirubin and the graphene being 3.3 Å. The adsorption energy of bilirubin was calculated as −48.4 kcal/mol, which suggests an energetically favorable adsorption process. We further analyzed the change in the total density of states (DOS) of graphene when modified with bilirubin and biliverdin, which is the reduced form of bilirubin (Figure 3). The DFT results show that pristine graphene is a zero-gap semiconductor, and its DOS around the Fermi level does not change considerably with the adsorption of bilirubin. More detailed analysis shows two molecular states corresponding to bilirubin 0.72 eV below the Fermi level and 1.25 eV above the Fermi level, which indicates that there is no thermally induced charge transfer between DOS of graphene and bilirubin. This is consistent with the analysis of the Bader charge on the support and adsorbed bilirubin, which confirms that there is a negligible charge transfer between graphene and bilirubin in its oxidized state. When bilirubin is in its reduced form, however, calculated DOS shows a discrete molecular level at the Fermi level that corresponds to a molecular state of biliverdin. Interaction between DOS of graphene and this state induces a charge transfer of −0.3e from the conduction band of graphene to biliverdin. This result shows that bilirubin in its reduced state has a higher affinity for electrons than the graphene, and we can expect some electron density to be transferred from the support to biliverdin even at 0 V vs SHE. To better understand the interfacial charge distribution between graphene and bilirubin in a real system, we calculated the difference in the charge density of graphene modified with bilirubin when the system is neutrally and positively charged. The results are shown in Figure 4 and illustrate how charge density on graphene or bilirubin changes when electrons are transferred from the support to the enzyme during the ORR performed by BOx. Bader charge analysis shows that when one

Figure 4. Upper: difference in the charge density between the neutral system (graphene with bilirubin adsorbed) and the positively charged system; sideways and top view. One unit cell is shown. Lower: HOMO and LUMO orbitals of bilirubin.

electron is removed from the system, the charge on bilirubin changes to +0.12e, while the charge on graphene changes to +0.88e. When two electrons are removed from the system, this induces a change of +0.42e on bilirubin and +1.58e on graphene. Difference in the charge density between the neutral and positively charged system also shows that the extraction of electrons induces a larger change in the charge density of the support. However, when electrons are removed from the system, some electron density will be removed from a HOMO orbital of bilirubin as well. Namely, the change in the electron density on the adsorbed bilirubin corresponds to the shape of its HOMO orbital. On the basis of these results, we can, thus, conclude that the transfer of electrons from the support modified with bilirubin to the enzyme will induce a delocalized change in the charge density across both the support and the modifier molecule. 3.3. Interaction between Bilirubin and BOx. We further studied the interaction between bilirubin and BOx by performing a series of docking simulations of bilirubin to BOx with AutoDock Vina. In the first set of simulations, the dockings were performed with both rigid and flexible bilirubin to rigid BOx. The geometries that correspond to the most favorable docking positions of bilirubin are shown in Figure 5. Binding affinities of rigid bilirubin and flexible bilirubin were determined as −7.6 and 7.5 kcal/mol. In addition, analysis of the obtained binding positions of bilirubin relative to the van der Waals surface of the enzyme reveals that in both cases bilirubin binds to the surface of the enzyme. The distance between bilirubin and T1 Cu atom was determined as 12.4 and 3637

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

Table 1. Summary of Docking of Bilirubin to BOx Obtained with Different Models Using AutoDock Vina

rigid pocket/rigid substrate rigid pocket/flexible substrate flexible pocket/rigid substrate flexible pocket/flexible substrate

binding energy (kcal/mol)

bilirubin−Cu distance (Å)

−7.6 −7.5

12.4 12.3

−10.1

5.7

−7.8

7.5

as 5.7 and 7.5 Å for rigid and flexible bilirubin, respectively. In contrast to the case in which BOx was treated as rigid, simulations with the flexible pocket result in binding of bilirubin deeper in the enzyme’s pocket. Namely, the distance between T1 Cu atom and bilirubin decreases from 12.3 to 5.7 Å. The analysis of the van der Waals surface of the binding pocket reveals that the binding pocket is very narrow before the binding of bilirubin and requires rearrangements in order for the substrate to position itself closer to T1 Cu (Figure S1 in Supporting Information). The opening of the pocket can be mainly attributed to the changes in the position of three amino acids on the surface of the enzyme, which include TRP200, ASN197, and THR306. Indole group of TRP200, for example, rotates around the Cβ−Cγ bond for almost 180° (Figure S2). The difference between rigid and “flexible” binding pocket results, therefore, reflects the dynamic nature of substrate docking. As was previously suggested, the pocket of BOx has to flex open prior to the substrate binding.5 To validate the results obtained with AutoDock Vina, we also performed a DFT study of the binding of bilirubin to the pocket of BOx. The model system included only the amino acids in the binding pocket, which were terminated with hydrogen atoms. Bilirubin was then placed into the pocket, and the whole structure was allowed to relax. The binding energy of bilirubin was calculated as −23.96 kcal/mol on the B3LYP/631G level of theory, and the obtained optimized structure of bilirubin in the binding pocket of BOx is shown in Figure 7. The comparison between DFT and AutoDock results shows that the binding configuration of rigid bilirubin to flexible BOx, obtained using the docking code, is very similar to the binding configuration of bilirubin obtained with DFT (Figure 8). There are some differences in the position of the amino acids that are situated deeper in the binding pocket such as TRP433 and ASN459, but this could be attributed to the differences in the model used in two types of calculations. In addition, the DFT results show that two of the amino acids additionally stabilize bound bilirubin in BOx by forming hydrogen bonds with it. ASN197 makes a hydrogen bond with the imino group of bilirubin, and TRP200 makes a hydrogen bond with the carboxyl group of bilirubin. The N−H···O distance between imino group of bilirubin and ASN197 is determined as 2.88 Å, and the C−H···O distance between TRP200 and carboxyl group of bilirubin is determined as 2.89 Å. The analysis of the DFT calculations also shows that bilirubin bound in the pocket of BOx is only 3.5 Å away from the nitrogen atom of HIS398, which is directly coordinated to Cu. The short distance between bilirubin and HIS398 could imply that this amino acid is involved in the electron transfer from the bilirubin to T1 Cu atom. Namely, the distance between HIS398 and T1 Cu is determined as 1.95 Å, and the electron transfer could proceed via the substrate−HIS398−Cu pathway. One additional

Figure 5. Upper: interaction of bilirubin with BOx as obtained using AutoDock Vina with a rigid BOx and (a) rigid bilirubin and (b) flexible bilirubin (yellow: bilirubin; cyan: amino acids in the experimentally determined binding pocket; pink: Cu atoms). Lower: binding of bilirubin to the van der Waals surface of BOx with the mapped electrostatic potential of the enzyme.

12.3 Å for rigid and flexible bilirubin, respectively. In the second set of simulations, the docking simulations were performed with both rigid and flexible bilirubin to BOx with a number of amino acids in the binding pocket treated as flexible. These included ASN197, SER198, TRP200, SER231, SER233, MET273, THR305, ASP306, TRP361, THR397, HIS398, TRP433, ASN459, and HIS462. The geometries of the most favorable binding positions of rigid and flexible bilirubin to BOx with the flexible pocket are shown in Figure 6. In this case, the

Figure 6. (a) Binding of rigid bilirubin to flexible bilirubin oxidase. (b) Binding of flexible bilirubin to flexible bilirubin oxidase as obtained using AutoDock Vina (yellow: bilirubin; cyan: flexible amino acids; pink: T1 Cu center).

binding affinity of rigid bilirubin was determined as −10.1 kcal/ mol while the lowest binding affinity obtained in the case of flexible bilirubin was −7.8 kcal/mol. The results of the docking also show that rigid bilirubin situates itself closer to the T1 Cu atom than flexible bilirubin (Table 1). Namely, the shortest distance between nonhydrogen atom of bilirubin and T1 Cu atom was determined 3638

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

Figure 7. Binding of bilirubin (yellow) in the pocket of bilirubin oxidase as obtained using the B3LYP/6-31G level of theory.

distance of the substrate relative to the support once the substrate on the support is defined as a docking ligand. In order to correct for this limitation, we had to redefine the way the docking simulations were performed. Therefore, bilirubin was first docked to BOx. After that, the graphene sheet was docked to the enzyme with bound bilirubin. In order to validate the obtained results, the outputs of the docking of the support to the enzyme with the bound substrate were finally used as inputs to perform the docking of the substrate adsorbed on the support to the enzyme. As expected by the experiment, this sequence of docking simulations resulted in the interaction of BOx with bilirubin (Figure 9). The binding affinity of bilirubin

Figure 8. Comparison between DFT (red) and AutoDock Vina results (blue) for binding of bilirubin in the pocket of bilirubin oxidase.

argument for this conclusion is the fact that HIS398 and HIS462 are highly conserved between MCOs from different organisms, which confirms their crucial role in the functioning of MCOs. 3.4. Interaction between the Support, Bilirubin, and BOx. First, bilirubin adsorbed on a graphene sheet was docked to BOx. The DFT optimized structure of bilirubin adsorbed on the graphene sheet shown in Figure 2b was used as the docking ligand. Because of the inability of the docking code to account for the periodicity of the support, the infinite graphene sheet was terminated and the support was treated as a finite graphene sheet with the dimensions of 34.2 Å × 41.3 Å. All docking simulations performed this way, by treating either bilirubin or BOx as rigid or flexible, resulted in the enzyme binding to the support exclusively (Figure S3). These results do not agree with our experimental findings because they suggest that the surface modification with bilirubin has no influence on the generated current density compared to the unmodified BOx cathode. This finding is a result of docking code’s inability to change the

Figure 9. Binding of bilirubin adsorbed on the support to BOx as obtained using AutoDock Vina (a) rigid and (b) flexible bilirubin (yellow: bilirubin; gray: graphene support; cyan: flexible amino acids; pink: Cu atom).

on the support was determined as −30.0 and −26.6 kcal/mol for rigid and flexible bilirubin, respectively. Furthermore, the analysis of the distance between bilirubin and the support or T1 Cu atom of BOx shows that bilirubin positions itself close to both the support and T1 Cu atom. Namely, in the case of rigid bilirubin the shortest distance between non-hydrogen atom of bilirubin and the support is 3.7 Å, while the distance between bilirubin and Cu atom is 5.6 Å. In the case of flexible bilirubin, the shortest distance between non-hydrogen atom of bilirubin and the support is 3.7 Å, while the distance between bilirubin 3639

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

the T1 Cu. Our results further reveal highly dynamic interaction between the enzyme, its substrate, and the support material. Namely, adsorption of bilirubin induces rearrangements in the enzyme’s binding pocket and changes the way bilirubin interacts with the surface of the electrode. Our application of the computational methods for the understanding of the support−modifier−enzyme interactions illustrates how computational approaches can be used as a valuable tool in a rational design of bioelectrodes for efficient biofuel cell applications. Docking simulations can be of particular relevance because they offer an extremely fast and efficient way for identifying chemical structures that will lead to optimal interactions on the support−biocatalyst interface. This in turn can result in smart selection of surface modification procedures that can ensure stable enzyme immobilization and enhanced electrode performance by favorable enzyme orientation and/or facilitated interfacial electron transfer.

and Cu atom is 7.6 Å. The distance of T1 Cu atom from the support is 16.3 and 16.4 Å in the case of rigid and flexible bilirubin, respectively. Figure 10 shows the difference in the binding of rigid bilirubin to the support before and after its interaction with

Figure 10. Difference in the binding of rigid bilirubin to the support before and after its interaction with BOx.

■

BOx. The circled area denotes the largest change in the position of the molecule relative to the support before and after the interaction with the enzyme. As can be seen, the distance of the central methylene bridge in bilirubin and the graphene surface increases from 3.8 to 5.6 Å, but the closest distance between bilirubin and the support changes only slightly from 3.3 to 3.7 Å. The results show that the positioning of bilirubin on the enzyme−support interface depends on both the interactions with the support and the enzyme. In conclusion, the results of DFT and docking simulations allow us to understand the observed enhancement in the generated current densities in BOx oxygen reduction cathode modified with bilirubin as compared to an unmodified electrode. On the basis of the calculated interaction energies and the analysis of the bilirubin’s positioning on the support− enzyme interface, we hypothesize that bilirubin serves as a geometric and electronic extension of the carbonaceous support. Because of beneficial positioning of bilirubin toward both the support and the enzyme, the modification of the support with bilirubin decreases the distance between the support and T1 Cu atom of BOx. As such, the presence of bilirubin on the electrode’s surface increases the electron transfer rate between the support and T1 Cu and results in a more efficient inlet of electrons to T1 center. Our results also suggest that histidine coordinated to T1 Cu could participate in the electron transfer between the modified support and the T1 Cu atom due to its bridging position between bilirubin and T1 Cu atom of the enzyme. We also propose that ASN197 participates in the proton transfer, which accompanies electron transfer when bilirubin is oxidized to biliverdin. Namely, ASN197 forms a hydrogen bond with imine nitrogen of bilirubin and could play an important role in the oxidation of bilirubin by BOx.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b01616. Three figures illustrating (1) van der Waals surface of the binding pocket of bilirubin to bilirubin oxidase before (crystal structure) and after the binding with bilirubin (obtained using AutoDock Vina), (2) the differences in the position of amino acids in the binding pocket of bilirubin to bilirubin oxidase before and after binding of bilirubin, and (3) docking of rigid and flexible bilirubin adsorbed on the support to bilirubin oxidase obtained by docking of bilirubin optimized on the support without the presence of the enzyme (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail [email protected]; Tel 505-277-2640 (P.A.). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by US DOD, ARO-Multi-University Research Initiative Grant W911NF-14-1-0263 to University of Utah. VASP license was provided by Theoretical division, LANL, which is supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC52-06NA25396. Computational work was performed using the computational resources of LANL, CNMS, sponsored at Oak Ridge National Laboratory by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy and NERSC, supported by the Office of Science of the U.S. Department of Energy under Contract DE-AC02-05CH1123. This paper has been designated LA-UR-16-20777.

4. CONCLUSIONS The surface modification of BOx based oxygen reduction cathode with enzyme’s natural substrate, bilirubin, induces an increase in open circuit potential and enhances the generated current densities of the cathode. The results of density functional theory and docking simulations confirm that this modification procedure is beneficial for positioning of BOx closer relative to the support. Bilirubin not only provides optimal orientation of the enzyme with its T1 Cu atom toward the support but also facilitates the interfacial electron transfer by decreasing the distance between the electrode surface and

■

REFERENCES

(1) Opallo, M.; Bilewicz, R. Recent Developments of Nanostructured Electrodes for Bioelectrocatalysis of Dioxygen Reduction. Adv. Phys. Chem. 2011, 2011, 1−21. (2) Calabrese Barton, S.; Gallaway, J.; Atanassov, P. Enzymatic Biofuel Cells for Implantable and Microscale Devices. Chem. Rev. 2004, 104, 4867−1−4886. (3) Minteer, S. D.; Liaw, B. Y.; Cooney, M. J. Enzyme-Based Biofuel Cells. Curr. Opin. Biotechnol. 2007, 18, 228−34.

3640

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641

Article

The Journal of Physical Chemistry B

AnodesMediation or Orientation Effect. J. Am. Chem. Soc. 2015, 137, 7754−7762. (23) Frisch, M. J.; et al. Gaussian 09, Revision B.01; Gaussian, Inc.: Wallingford, CT, 2009. (24) Dion, M.; Rydberg, H.; Schröder, E.; Langreth, D. C. Phys. Rev. Lett. 2004, 92, 246401. (25) Klimeš, J.; Bowler, D. R.; Michaelides, A. J. Phys.: Condens. Matter 2010, 22, 022201. (26) Blöchl, P. E. Projector Augmented-Wave Method. Phys. Rev. B: Condens. Matter Mater. Phys. 1994, 50, 17953−17979. (27) Kresse, G.; Joubert, J. From Ultrasoft Pseudopotentials to the Projector Augmented-Wave Method. Phys. Rev. B: Condens. Matter Mater. Phys. 1999, 59, 1758−1775. (28) Kresse, G.; Hafner, J. Ab Initio Molecular Dynamics for Liquid Metals. Phys. Rev. B: Condens. Matter Mater. Phys. 1993, 47, 558−561. (29) Kresse, G.; Hafner, J. Ab Initio Molecular-Dynamics Simulation of the Liquid-Metal−Amorphous-Semiconductor Transition in Germanium. Phys. Rev. B: Condens. Matter Mater. Phys. 1994, 49, 14251−14269. (30) Kresse, G.; Furthmüller, J. Efficiency of Ab-Initio Total Energy Calculations for Metals and Semiconductors Using a Plane-Wave Basis Set. Comput. Mater. Sci. 1996, 6, 15−50. (31) Kresse, G.; Furthmüller, J. Phys. Rev. B: Condens. Matter Mater. Phys. 1996, 54, 11169. (32) Monkhorst, H. J.; Pack, J. D. Special Points for Brillouin-Zone Integrations. Phys. Rev. B: Condens. Matter Mater. Phys. 1976, 13, 5188−5192. (33) Tang, W.; Sanville, E.; Henkelman, G. J. Phys.: Condens. Matter 2009, 21, 084204. (34) Sanville, E.; Kenny, S. D.; Smith, R.; Henkelman, G. J. Comput. Chem. 2007, 28, 899−908. (35) Henkelman, G.; Arnaldsson, A.; Jónsson, H. Comput. Mater. Sci. 2006, 36, 354−360. (36) Trott, O.; Olson, A. J. Autodock Vina: Improving the Speed and Accuracy of Docking with a New Scoring Function, Efficient Optimization, and Multithreading. J. Comput. Chem. 2009, 31, 455− 461. (37) Morris, G. M.; Huey, R.; Lindstrom, W.; Sanner, M. F.; Belew, R. K.; Goodsell, D. S.; Olson, A. J. Autodock4 and Autodocktools4: Automated Docking with Selective Receptor Flexibility. J. Comput. Chem. 2009, 30, 2785−91. (38) Sanner, M. F. Python: A Programming Language for Software Integration and Development. J. Mol. Graphics Modell. 1999, 17, 57− 61. (39) Humphrey, W.; Dalke, A.; Schulten, K. Vmd - Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (40) Taylor, R.; Kennard, O.; Versichel, W. Geometry of the N-H··· OC Hydrogen Bond. 2. Three-Center (“Bifurcated”) and Four Center (“Trifurcated”) Bonds. J. Am. Chem. Soc. 1984, 106, 244−248. (41) Rai, A. K.; Rai, S. B.; Rai, D. K.; Singh, V. B. Spectroscopic Studies and Normal Coordinate Analysis of Bilirubin. Spectrochim. Acta, Part A 2002, 58, 2145−2152.

(4) Mizutani, K.; et al. X-Ray Analysis of Bilirubin Oxidase from Myrothecium Verrucaria at 2.3 Angstrom Resolution Using a Twinned Crystal. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2010, 66, 765−770. (5) Cracknell, J.; McNamara, T.; Lowe, E.; Blanford, C. Bilirubin Oxidase from Myrothecium Verrucaria: X-Ray Determination of the Complete Crystal Structure and a Rational Surface Modification for Enhanced Electrocatalytic O-2 Reduction. Dalton Transactions 2011, 40, 6668−6675. (6) Solomon, E. I.; Sundaram, U. M.; Machonkin, T. E. Multicopper Oxidases and Oxygenases. Chem. Rev. 1996, 96, 2563−2605. (7) Christenson, A.; Shleev, S.; Mano, N.; Heller, A.; Gorton, L. Redox Potentials of the Blue Copper Sites of Bilirubin Oxidases. Biochim. Biophys. Acta, Bioenerg. 2006, 1757, 1634−1641. (8) Barriere, F.; Ferry, Y.; Rochefort, D.; Leech, D. Targeting Redox Polymers as Mediators for Laccase Oxygen Reduction in a MembraneLess Biofuel Cell. Electrochem. Commun. 2004, 6, 237−241. (9) Chen, R. J.; Zhang, Y.; Wang, D.; Dai, H. Noncovalent Sidewall Functionalization of Single-Walled Carbon Nanotubes for Protein Immobilization. J. Am. Chem. Soc. 2001, 123, 3838−3839. (10) Lopez, R. J.; Babanova, S.; Ulyanova, Y.; Singhal, S.; Atanassov, P. Improved Interfacial Electron Transfer in Modified Bilirubin Oxidase Bio-Cathodes. ChemElectroChem. 2014, 1, 241−248. (11) Ramasamy, R. P.; Luckarift, H. R.; Ivnitski, D. M.; Atanassov, P.; Johnson, G. R. High Electrocatalytic Activity of Tethered Multicopper Oxidase−Carbon Nanotube Conjugates. Chem. Commun. 2010, 46, 6045−6047. (12) Babanova, S.; Matanovic, I.; Atanassov, P. Quinone-Modified Surfaces for Enhanced Enzyme-Electrode Interactions in Pyrroloquinoline-Quinone-Dependent Glucose Dehydrogenase Anodes. ChemElectroChem 2014, 1, 2017−2028. (13) Riklln, A.; Katz, E.; Willner, I.; Stocker, A.; Buckmann, A. Improving Enzyme-Electrode Contacts by Redox Modification of Cofactors. Nature 1995, 376, 672−675. (14) Chakraborty, S.; Babanova, S.; Rocha, R. C.; Desireddy, A.; Artyushkova, K.; Boncella, A. E.; Atanassov, P.; Martinez, J. S. A Hybrid DNA-Templated Gold Nanocluster for Enhanced Enzymatic Reduction of Oxygen. J. Am. Chem. Soc. 2015, 137, 11678−11687. (15) Ramirez, P.; Mano, N.; Andreu, R.; Ruzgas, T.; Heller, A.; Gorton, L.; Shleev, S. Direct Electron Transfer from Graphite and Functionalized Gold Electrodes to T1 and T2/T3 Copper Centers of Bilirubin Oxidase. Biochim. Biophys. Acta, Bioenerg. 2008, 1777, 1364− 1369. (16) Gutiérrez-Sánchez, C.; Jia, W.; Beyl, Y.; Pita, M.; Schuhmann, W.; De Lacey, A.; Stoica, L. Enhanced Direct Electron Transfer between Laccase and Hierarchical Carbon Microfibers/Carbon Nanotubes Composite Electrodes. Comparison of Three Enzyme Immobilization Methods. Electrochim. Acta 2012, 82, 218−223. (17) Brocato, S.; Lau, C.; Atanassov, P. Mechanistic Study of Direct Electron Transfer in Bilirubin Oxidase. Electrochim. Acta 2012, 61, 44− 49. (18) Reda, T.; Hirst, J. Interpreting the Catalytic Voltammetry of an Adsorbed Enzyme by Considering Substrate Mass Transfer, Enzyme Turnover, and Interfacial Electron Transport. J. Phys. Chem. B 2006, 110, 1394−1404. (19) Blanford, C. F.; Heath, R. S.; Armstrong, F. A. A Stable Electrode for High-Potential, Electrocatalytic O2 Reduction Based on Rational Attachment of a Blue Copper Oxidase to a Graphite Surface. Chem. Commun. 2007, 1710−1712. (20) Giroud, F.; Minteer, S. D. Anthracene-Modified Pyrenes Immobilized on Carbon Nanotubes for Direct Electroreduction of O2 by Laccase. Electrochem. Commun. 2013, 34, 157−160. (21) Ulyanova, Y.; Babanova, S.; Pinchon, E.; Singhal, S.; Matanovic, I.; Atanassov, P. Effect of Enzymatic Orientation through the Use of Syringaldazine Molecules on Multiple Multicopper Oxidase Enzymes. Phys. Chem. Chem. Phys. 2014, 16, 13367. (22) Babanova, S.; Matanovic, I.; Chavez, M. S.; Atanassov, P. Role of Quinones in Electron Transfer of PQQ−Glucose Dehydrogenase 3641

DOI: 10.1021/acs.jpcb.6b01616 J. Phys. Chem. B 2016, 120, 3634−3641