Molecular Simulations of Cytochrome c Adsorption on a Bare Gold

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Molecular Simulations of Cytochrome c Adsorption on a Bare Gold Surface: Insights for the Hindrance of Electron Transfer Chunwang Peng, Jie Liu, and Jian Zhou J. Phys. Chem. C, Just Accepted Manuscript • Publication Date (Web): 14 Aug 2015 Downloaded from http://pubs.acs.org on August 14, 2015

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The Journal of Physical Chemistry

Molecular Simulations of Cytochrome c Adsorption on a Bare Gold Surface: Insights for the Hindrance of Electron Transfer

Chunwang Peng, Jie Liu, Jian Zhou*

School of Chemistry and Chemical Engineering, Guangdong Provincial Key Lab for Green Chemical Product Technology, South China University of Technology, Guangzhou, Guangdong, 510640, P. R. China

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ABSTRACT: Experimental studies have shown that the adsorption of horse heart Cytochrome c (Cyt-c) on a bare gold surface leads to the hindrance of electron transfer (ET). The underlying mechanism remains controversial. In this work, the adsorption orientation and conformation of Cyt-c on Au(111) surface were investigated by performing Monte Carlo and molecular dynamics simulations. The results show that the most favorable binding mode of Cyt-c to gold agrees with the results characterized by SEIRA spectroscopy. The adsorption is mainly contributed by the strong van der Waals interactions between the surface and residues that have long side chains, which leads to the helix A and Ω1 loop of Cyt-c being in contact with the surface and most of the α-helixes being nearly parallel to the surface. The native structure of Cyt-c is well preserved during the adsorption and only the flexible Ω1 loop and the N-terminal show a relatively larger mobility. The hindrance of ET is ascribed to the confined rotation of the heme prosthetic group and the farther positioning of the central iron to the surface (about 12.9 Å).

KEYWORDS: Molecular simulation, Au(111), protein adsorption, protein orientation, protein conformation

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1. INTRODUCTION The direct electron transfer (ET) between redox protein and the electrode surfaces plays an important role in bioelectrocatalysis, biosensors, enzymatic biofuel cells and biochips1-6. Cytochrome c (Cyt-c), a water-soluble redox protein residing in the inner mitochondrial membrane, is often used as a typical representative in the study of the structure, thermodynamics, kinetics and direct ET reactions of hemeprotein to reveal the ET mechanisms inside and outside the membranes of living cells. The two kinds of widely studied Cyt-c are extracted from yeast and horse heart, and the adsorption of them on the bare gold surface results in different ET properties. The former can be covalently attached to bare gold surfaces through the cysteine residue (Cys102) to form a stable monolayer and reversible redox peaks can be observed in cyclic voltammetry (CV) measurements.7-10 However, there is no cysteine at the surface for the horse heart Cyt-c and no observable ET can be measured when adsorbed onto bare gold surfaces.11-15 This has been disputably ascribed to the formation of aggregates12, surface blocking by progressive adsorption of inactive molecules13, adsorption-induced unfolding or even denaturation of the protein14, 16, or the conformation and orientation changes of the heme15, 17. In order to explore the mechanism of horse heart Cyt-c (abbreviated as Cyt-c in the following, unless specified) adsorption on bare gold surfaces, many efforts have been made. Among them, two studies have been used to detect its adsorption orientation by modern spectroscopic techniques. Yu and Golden17 applied surface-enhanced Raman scattering (SERS) to probe the orientations of Cyt-c on gold nanohole arrays coated with or without oppositely charged self-assembled monolayers (SAMs). The results indicated that the heme

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group was near and almost vertical to the negatively charged surface while far away from the positively charged surface.17 However, Cyt-c had a random or relatively flat orientation on the bare gold nanohole surface.17 Lin et al.15 probed the adsorption of Cyt-c on a bare gold surface by surface-enhanced infrared absorption (SEIRA) spectroscopy. The results showed that Cyt-c kept its native structure when adsorbed on the bare gold, with most of the α-helixes running parallel to the surface.15 They ascribed the hindrance of ET to the adsorption orientation and suppressed rotation of the adsorbed Cyt-c. However, the adsorption orientations of Cyt-c on bare gold surface characterized by SERS and SEIRA are quite different. What is the cause of the hindrance of ET? Although some experiments can provide structural, electronic and dynamic information simultaneously, in situ detection of the orientation and conformation changes of proteins emerging at the molecular level is still technically challenging.15 Molecular simulations are very suitable to study the behavior of protein adsorption on surfaces at the molecular level.18 So far, a few molecular dynamics (MD) simulation studies of Cyt-c adsorption on solid surfaces have been reported.18-25 Zhou et al.18 studied the adsorption of Cyt-c on carboxyl-terminated SAMs (COOH-SAM) by performing Monte Carlo and MD simulations. They showed that the heme ring was near and almost vertical to the COOH-SAM surface.18 Alvarez-Paggi et al.21-23 employed MD simulations and electron pathway analyses to investigate the ET properties of Cyt-c adsorbed on gold functionalized by COOH-SAM. Their findings demonstrated that the ET between the adsorbed Cyt-c and the electrode was determined by the interplay between protein dynamics and tunneling probabilities.21 Hung et al.24 investigated the adsorption of Cyt-c on monolayer-protected metal nanoparticles (MPMNs) through a combined 4


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coarse-grained and all-atom simulation approach. Their results showed that the heme ring was near and almost vertical to the methyl-terminated SAMs while it is nearly parallel to the hydroxyl-terminated SAMs. Recently, Xie et al.25 employed MD simulations to explore the influence of applied electric fields on the adsorption of Cyt-c onto zwitterionic phosphorylcholine SAMs. However, simulation study of horse heart Cyt-c adsorption on a bare gold surface has not been reported yet. Gold is widely used in the field of bioelectrochemistry due to its good biocompatibility, electrical conductivity, corrosion resistance, and unique catalytic properties26. Meanwhile, the Au(111) surface is one of the most commonly featured facets of gold nanoparticles (AuNPs).27 Details of Cyt-c on Au(111) surface can be used as the basis for the investigation of its adsorption on AuNPs, which are found to facilitate its ET28-29. For simulations of gold, traditional force fields only consider the van der Waals (vdW) interactions (UvdW), which are not accurate enough to describe the adsorption of biomolecules on gold surface. The Gold-Protein (GolP) force field30 was developed, which was compatible with OPLS/AA to describe the interaction between biomolecules and Au(111) surfaces in aqueous solution. This force field takes into account the vdW interaction, polarization of gold, chemisorption and conjugated (π) systems. Moreover, virtual sites are introduced to reproduce the real adsorption sites. Recently, it has been used to study the adsorption of yeast Cyt-c on the Au(111) surface by MD simulations to investigate the structural relaxation and its role in ET between the protein and the surface.31 Meanwhile, they have extended the GolP force field to be compatible with CHARMM force field and obtained the GolP-CHARMM force field32.

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In this work, the Parallel Tempering Monte Carlo (PTMC) algorithm33 will be employed to obtain the preliminary orientation of the adsorbed protein with global minimum energy in one simulation. It has been successfully used in some of our previous works34-39 to quickly obtain the preferential adsorption orientations. With these preliminarily optimized orientations from PTMC as the initial orientations, MD simulations were performed by using the GolP-CHARMM force field32 to explore the adsorption behavior of Cyt-c on Au(111) surface. Experimental results15, 40-41 have shown that the adsorption of Cyt-c on COOH-SAM facilitates its ET between the heme group and the electrode surfaces. For comparison, simulations of Cyt-c on COOH-SAM will also be conducted. The adsorption conformation and orientation of Cyt-c on the two surfaces will be studied in detail. Finally, the possible mechanism for the hindrance of ET for Cyt-c adsorbed on bare gold surfaces will be discussed. 2. MATERIALS AND METHODS 2.1. Protein and Surfaces Cyt-c. The crystal structure of horse heart Cyt-c42 (PDB-ID: 1HRC) obtained from the RCSB (www.rcsb.org) is used in our simulations. It consists of 104 residues and a prosthetic group (heme), with five α-helixes and two anti-parallel β-sheets (Figure 1). The overall structure is approximately spherical with a diameter of about 3.4 nm. The heme iron is coordinated with a histidine (His18) and a methionine (Met80), and two vinyl groups of the heme ring are covalently bonded to two cysteines (Cys14 and Cys17) via thioether bonds.43 The protein was studied at physiological conditions, with lysine and arginine residues being protonated, while aspartic acid and glutamic acid residues together with the C-terminus being 6


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deprotonated.18 The N-terminus was acetylated as it presents in the crystal structure. Hydrogen atoms were added by the pdb2gmx tool of GROMACS 4.5.4 package44. Heme was treated in oxidation state42 (consistent with the crystal structure), the charge distribution and force field parameters for heme and its linkage with the peptide chain were adopted from Autenrieth et al.’s work.43 The protein contains 1749 atoms bearing a net charge of +7e. The potential parameters for Cyt-c are adopted from the CHARMM27 force field45.

Figure 1. Secondary structure assignment of horse heart Cyt-c and illustration of the orientation angles. Cyt-c is displayed in NewCartoon mode and the heme is represented in Licorice mode. The three arrows: blue (n), black (m) and red (e) show the direction of surface normal, heme plane normal and the dipole, respectively. Au(111). Crystal structure of gold came from the Materials Studio software and the lattice parameter was set to 2.93 Å32. The Au(111) surface containing five atomic layers with a dimension of 71.05×70.32 Å2 was constructed. Each gold atom was replaced with a dipole made up by two opposite image charges (± 0.3 e) and two virtual sites were introduced for each real gold surface atom.32 The vdW interaction of gold surface atoms with a biomolecule is described indirectly through the virtual interaction sites only.30, 32 7



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The GolP-CHARMM force field32 takes into account the dynamic polarization of gold atoms, chemisorption, and the interaction of sp2 hybridized carbon with gold atoms. The potential energy function32 for the interaction between the adsorbate and the gold surface is: π VAutot− X = VAuim− X + VAuvdW− X + VAuchemisorb + VAu −X −X ,

(1)

where VAuim− X is the electrostatic interaction between the adsorbate and the image charges π induced upon adsorption, and VAuvdW− X , VAuchemisorb , and VAu are the vdW, chemisorbing −X −X

species, and π electron interactions between the adsorbate and gold. The force field can be used to study peptide adsorption on both the Au(111) and Au(100) surfaces in aqueous solution, and can give a deep understanding of small molecule facet selectivity in vacuo and reproduce the energetic and spatial trends as those observed in the DFT calculations.32 COOH-SAM. The (

3 × 3 )R30° lattice structure46-47 was employed for SAM of

S(CH2)10COOH in our MD simulations. The surface contains 168 thiol chains and the dimension is 59.94×60.56 Å2. Among them, 12 chains are deprotonated, resulting in a surface charge density (SCD) of 0.05C/m2.35 All sulfur atoms in the SAM were kept fixed during the simulations. The potential parameters for the SAM are adopted from the CHARMM force field for lipids.48 2.2. PTMC Simulations The protein is simulated using a united-residue model, that is, each amino acid is treated as a sphere centered at its α-carbon, which well keeps the basic structure of the protein.49 The surface is taken to be flat. The parameters are adopted from our previous works.33, 35, 49 The protein is kept rigid during PTMC simulations. N replicas are computed in parallel, with each of them simulated in the NVT ensemble and at a different temperature T.37 In this work, each 8


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replica was calculated in a box with a dimension of 10×10×10 nm3. Firstly, Cyt-c was placed in the box center. Then, by translating and rotating the protein around its mass center, 40,000,000 MC cycles were performed with the first half for equilibrium and the second half for production. By adjusting the displacement of each move, an acceptance ratio of 0.5 was ensured. Through uninterruptedly attempting, six replicas were chosen, and temperatures are 300, 350, 450, 550, 650 and 750 K.37 They can insure sufficient energy overlap between adjacent replicas so that acceptable configuration exchanges can be obtained for each system.37 The exchanges are carried out every 500 cycles. Details are similar to that described in our previous works.33, 35, 37, 49 The most dominant orientations were chosen as the starting points for further MD simulations. 2.3. MD Simulations The most dominant orientations obtained from PTMC simulations were selected as the initial points. Water molecules were filled in a box of 71.05×70.32×76 Å3 and 59.94×60.56 ×80 Å3 for the Au(111) and COOH-SAM, respectively. The TIP3P model50 was used in this work. Chloride and sodium ions were added to neutralize the system and the ionic strength was set to 0.01 M. The simulations were carried out in a NVT ensemble and the time step is 2 fs. The temperature was maintained at 300 K via a Nose-Hoover thermostat51 every 0.5 ps. The LINCS algorithm52 were employed to constrain bonds containing hydrogen atoms. A switched potential was adopted to calculate the nonbonded interactions with a switching function between 9 Å and 10 Å. Electrostatic interactions were calculated in 3dc geometry53 by employing the Particle Mesh Ewald (PME) method. Two-dimensional periodic boundary conditions were used with two hard walls placed at the two ends (in z direction) of the 9



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simulation box37, and the scaling factor for the z direction for Ewald summation was 3, as implemented in the GROMACS 4.5.4 package44. The cutoff distance of the electrostatic interactions was set to be 11 Å. Firstly, minimize the systems using the steepest descent method to remove inappropriate geometry or steric overlap. Then, a NVT equilibration with duration of 100 ps was performed for each system to equilibrate the water molecules and ions around the protein. During the NVT simulations, the positions of heavy atoms of the protein were restrained. Finally, each system went through a 100-ns MD simulation. The trajectories were visualized via the Visual Molecular Dynamics (VMD) program54. 2.4. Orientation Characterization The orientation angle is applied to quantitatively characterize the orientation of a protein on a surface.37 The dipole angle (θ)18, 35, 37 is defined as the angle between the directions of protein dipole (e) and the surface normal (n) (as shown in Figure 1). The cosine value of this angle is used to characterize the orientation of adsorbed Cyt-c. For values of 1 or -1, the dipole is perpendicular to the surface; while it is parallel to the surface for the value of 0. Furthermore, the heme tilt angle (α)18, 21, defined as the angle between the heme plane normal and the surface normal, is also used to characterize the orientation of Cyt-c. The value of 0° or 180° implies that the heme is parallel to the surface, whereas the value of 90° indicates it is perpendicular to the surface. 3. RESULTS AND DISCUSSION The adsorption of Cyt-c on Au(111) was investigated by performing PTMC and MD simulations. For comparison, behaviors of Cyt-c in the bulk solution and on COOH-SAM were also studied. The interaction energies between Cyt-c and the surfaces, binding sites, 10


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orientation distribution and conformation of Cyt-c were analyzed from MD simulation trajectories. Finally, the possible mechanisms for the hindrance of ET for Cyt-c adsorbed on bare gold surfaces were discussed. The results are shown in Tables 1~2 and Figures 2~7. 3.1. Adsorption Orientation PTMC Simulations. PTMC simulations were carried out to obtain the preliminary orientations of Cyt-c adsorbed on the Au(111) surfaces. Considering that the binding of Cyt-c on gold may affect the polarization of the surface, a neutral and two weakly charged surfaces49 were chosen with SCDs of 0 and ± 0.006 C/m2, respectively. Orientation distributions of the dipole of Cyt-c on different surfaces obtained from PTMC simulations are shown in Figure 2. It can be seen that Cyt-c has a dominant conformation on each surface. The three dominant orientations O1 (0.941), O2 (0.129) and O3 (−0.544) for surfaces with SCDs of 0.006, 0 and −0.006 C/m2 are shown in Figure 3a-c. All the three most dominant orientations were selected as starting orientations for further MD simulations.

Figure 2. Orientation distributions of the dipole of Cyt-c on different surfaces by PTMC simulations. The blue, red and black lines correspond to the positively charged, negatively charged and neutral surfaces, respectively.

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To compare with the experimental results, two other different orientations have also been selected as the initial configurations for MD simulations. The SEIRA (shown in Figure 3d) and SERS (shown in Figure 3e) represent the orientations derived from SEIRA spectroscopy15 and SERS17 for Cyt-c on bare gold, respectively. As can be seen, the preferred O3 orientation (Figure 3c) is a little bit close to the SEIRA orientation (Figure 3d), while the O1 orientation (Figure 3a) is very close to that derived from SERS for Cyt-c on gold functionalized with amino-terminated SAMs17. MD Simulations. To explore the adsorption behavior of Cyt-c on Au(111) surface, five different systems were constructed. The three dominant orientations (O1, O2, and O3) derived from PTMC simulations along with two other orientations derived from SERS17 and SEIRA spectroscopy15 were selected as the initial orientations. All these simulations were performed for 100 ns. The final snapshots of Cyt-c adsorbed on Au(111) surface are shown in Figure 3f-j. For comparison, the O3 orientation was selected as the initial points for Cyt-c on COOH-SAM and the simulation was conducted for 100 ns also. It can be found that the final orientations for O3 (Figure 3h) and SERS (Figure 3j) are almost the same as that of SEIRA (Figure 3i), where most of the α-helixes of adsorbed Cyt-c are nearly parallel to the gold surface15. With long enough simulations, the O3 and SERS orientations may achieve a final adsorption as same as that of SEIRA. For the SERS initial orientation, a certain degree of orientation adjustment occurred during the simulation, which indicates that the orientation is not stable for Cyt-c on Au(111) surface.

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Figure 3. Initial and final configurations for MD simulations of Cyt-c adsorbed on Au(111) surface. (a)-(c) are the three preferred orientations from PTMC simulations; (d) and (e) are from experimental results; (f)-(j) are the corresponding configurations after MD simulations starting from (a)-(e). The red arrows indicate the direction of the dipole. Water and ions along with the dipole atoms and virtual sites of Au(111) surface are not shown for clarity. 3.2. Interaction energies To further investigate the most favorable binding mode and the underlying mechanisms for the different behaviors of Cyt-c on Au(111) and COOH-SAM, the interaction energies between Cyt-c and the surfaces were analyzed and the results are summarized in Table 1. The calculation of free energies for such a large system is too costly for the current resources. Furthermore, Cyt-c is one of the common hard proteins and it has little conformation change during the adsorption15, which will be discussed in Section 3.5. Therefore, the entropy contribution to the adsorption behavior is relatively small. Thus, we think that the calculation of interaction energies should be appropriate for the choice of the optimal binding mode. Here, the UvdW, Uele and Utot (Utot=UvdW+Uele) refer to the vdW, electrostatic and total interaction energies, respectively.37 To ascertain the equilibrium behavior, we have also calculated the 13



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evolution of the interaction energies between Cyt-c and the Au(111) surface (shown in Figure S1, Supporting Information), and the tilt angle of the heme group for Cyt-c adsorbed on the Au(111) and COOH-SAM surfaces (shown in Figure S2, Supporting Information) during the MD simulations. It is obvious that almost all these simulations have reached equilibrium except for the O2 orientation which has undergone a reorientation at time ~ 86 ns. It can be found from Table 1 that the interaction energy is the lowest (i.e., most favorable) for SEIRA orientation, which agrees with the experimental results characterized by SEIRA spectroscopy15. It can be seen that the vdW interaction (−472 kJ/mol) for Cyt-c on Au(111) is much stronger than the electrostatic interaction (−55 kJ/mol); while the electrostatic interaction (−548 kJ/mol) for Cyt-c on COOH-SAM is much stronger than the vdW interaction (−135 kJ/mol). Therefore, the adsorption of Cyt-c onto Au(111) and COOH-SAM are dominated by vdW interactions and electrostatic interactions, respectively. Table 1. Interaction energiesa between Cyt-c and the surfaces System

Evdw

Etot

O1

−389±21

−64±18

−453±32

O2

−252±27

−30±14

−282±28

O3

−309±14

−14±8

−323±17

SEIRA

−472±20

−55±19

−527±32

SERS

−338±12

−22±10

−360±17

O3

−135±34

−548±112

−683±123

Au(111)

COOH-SAM a

Eele

−1

The unit of the energies are kJ•mol .

3.3. Binding Sites To unravel the key residues accounted for the adsorption of Cyt-c on Au(111) surface, residues of Cyt-c in contact with the surface after the simulations reach equilibrium are picked out. For comparison, the adsorption configuration and contact residues for Cyt-c on 14


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COOH-SAM are also given. The results are shown in Figure 4. The residues containing atoms which are within 3.5 Å to the surface are thought to be in contact with the surface.31, 37

Figure 4. The most favorable orientations of Cyt-c adsorbed on Au(111) and COOH-SAM surfaces. The upper row and the lower row are for the side-view and top-view configurations, respectively. Residues less than 3.5 Å from the surface are represented in Licorice mode and colored by ResType. For top view, only the portion of Cyt-c near the surface is shown and the surfaces are shown in transparent mode. It can be seen from Figure 4 that the adsorption of Cyt-c on Au(111) surface leads to the helix A and Ω1 loop (residues 20-30)23 being in contact with the surface. Most of the contact residues (such as Gln12, Gln16, Lys25, His26 and Lys27) adsorb on the surface with their side chains being parallel to the surface in order to maximize the contact area. This greatly enhances the vdW interactions between Cyt-c and the Au(111) surface, which leads to a stronger adsorption. For COOH-SAM, the binding is mainly contributed by the C-terminal helix A, Ω3 loop and the small helix D. The contact lysines are consistent with previous experimental observations15,

55-56

and simulation results21. Adsorption of Cyt-c on 15



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COOH-SAM results in most of the α-helixes being nearly perpendicular to the surface. Meanwhile, the dipole direction of Cyt-c is also nearly vertical to the surface. This is in accord with previous works that protein orientation on a charged surface is dominated by its dipole direction.18, 33, 35, 37 To further investigate the residues responsible for adsorption, the interaction energies between contact residues of Cyt-c and the surfaces were calculated, and the results are summarized in Figure 5. In previous works, the adsorption of Cyt-c on the bare gold surface has been ascribed to the two cysteine residues (Cys14 and Cys17).14-15 However, these residues are almost imbedded in the pocket and are covalently linked to two vinyl groups of the heme. For comparison, the interaction energies between the cysteine residues (Cys14 and Cys17) and the gold surface were also calculated.

Figure 5. Interaction energies between contact residues of Cyt-c and the (a) Au(111) and (b) COOH-SAM surfaces It can be found from Figure 5a that residues with long side chains (such as Gln12, Gln16, Lys25, His26 and Lys27) are accounted for the strong vdW interactions between Cyt-c and the gold surface. However, the interactions between Cys14, Cys17 and the gold surface are negligible. Further calculations show that the distance (Z-direction) between the sulfur atoms

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of the two residues and gold surface is (1.03 ± 0.02) nm and (0.69 ± 0.03) nm, respectively. Thus, we believe that they may be not responsible for the binding of Cyt-c to the bare gold surface. In contrast, there is a cysteine residue (Cys102) at the surface of yeast cyt-c, thus it can be directly attached to the gold surface.7-10 From Figure 5b, we can deduce that the positively charged residues (Lys13, Lys72, Lys73, Lys86 and Lys87) play a leading role in the adsorption of Cyt-c on the negatively charged COOH-SAM. These residues contribute to about 89% of the total electrostatic interaction energies. 3.4. Orientation Distribution It has been proposed that in order to make electron transfer possible and fast, Cyt-c undergoes a rearrangement of the heme with respect to the electrode surface by changing its conformation or orientation.21, 40 However, this might be suppressed when Cyt-c is adsorbed on the bare gold surface.15 To investigate the orientation distributions of Cyt-c adsorbed on Au(111) and COOH-SAM surfaces, we use two different angles (dipole angle and heme tilt angle) to characterize them (as defined in section 2.4). The angle distributions were derived by statistical analysis of the last 20 ns MD trajectories for the optimal adsorption configurations of Cyt-c on the two surfaces. The results are displayed in Figure 6. To ascertain the convergence of these parameters, we have also calculated the evolution of the heme tilt angle (Figure S2, Supporting Information) and dipole direction of Cyt-c (Figure S3, Supporting Information) relative to the normal of the Au(111) and COOH-SAM surfaces during the MD simulations. It can be seen that both the dipole direction and the heme tilt angle for Cyt-c adsorbed on the Au(111) (for the optimal mode SEIRA) and COOH-SAM

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surfaces have reached equilibrium with only a little fluctuations around the equilibrium values.

Figure 6. Orientation distributions of Cyt-c adsorbed on Au(111) and COOH-SAM surfaces by MD simulations for (a) the dipole angle, and (b) heme tilt angle. As can be seen in Figure 6a, the protein dipole is almost vertical to the COOH-SAM surface, as revealed by the value of cosθ which is close to -1; while it is nearly parallel to the Au(111) surface. Moreover, Cyt-c has a narrower distribution of dipole angle on the COOH-SAM surface than that on Au(111) surface; while it has a wider distribution of heme tilt angle on the COOH-SAM surface than that on Au(111) surface. It has been recognized that the adsorption of a charged protein on an oppositely charged surface always leads to a relative narrow distribution of the dipole angle20, 35 and the variability of the dipole angle reduces with the increase of the SCD of the SAM18, 20. This is mainly due to the repositioning of side

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chains of the charged residues owing to the electrostatic interactions exerted by the charged surface. Therefore, it does not have a direct correlation with the protein orientation characterized by the heme tilt angle.20 Actually, due to the large mobility of the SAM surface, Cyt-c has a wider distribution of heme tilt angle when it adsorbs on the COOH-SAM surface. This suggests that Cyt-c adsorption on COOH-SAM provides more freedom for a repositioning movement of the heme than that on Au(111) surface. 3.5. Protein Conformation To investigate the structural stability of Cyt-c adsorbed on the surfaces, root mean square fluctuations (RMSF) of backbone atoms per residue were calculated, and the results are shown in Figure 7. Furthermore, the simulated structures of Cyt-c in bulk solution and on surfaces were superimposed on its crystal structure by VMD54. The results are also shown in Figure 7. It is obvious that no notable differences are observed between the simulated structures, which indicate that Cyt-c is in a stable conformation. The crystal structure of Cyt-c is well-kept when adsorbed on both surfaces, which agrees well with the experimental observations15, 40. It is evident that the Ω1 loop (residues 20-30) and Ω2 loop (residues 40-49) of Cyt-c have a higher mobility on Au(111) than that in the bulk solution and on COOH-SAM surface. The flexibility of those regions (especially the Ω1 loop) has been found in several simulation studies.21, 23, 57 These can be correlated with the disappearing of the h-bonded turn in the two segments and results in a larger flexibility of these residues.31 In addition, the N-terminal shows a bigger mobility than the other parts of Cyt-c. However, the fluctuation of the C-terminal is greatly restricted owing to the capping of the acetyl group.

19



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Figure 7. The mobility (RMSF) of backbone atoms per residue of Cyt c in bulk solution and on Au(111) and COOH-SAM surfaces. Inset: simulated structure of Cyt-c in bulk solution (cyan) and on Au(111) (yellow) and COOH-SAM (red) surfaces superimposed on its crystal structure (blue). To quantitatively describe the conformation changes of Cyt-c, root mean square deviation (RMSD) of the backbone atoms, radius of gyration (Rg), eccentricity and dipole moment of Cyt-c were calculated and the results were summarized in Table 2. With a careful comparison, we can find that the properties of Cyt-c in its crystal structure and in bulk solution are in excellent accordance with those calculated by Zhou et al.18. The RMSD values are all less than 2.0 Å suggesting that the secondary structure of Cyt-c is well kept when adsorbed on the surfaces and no partial denaturation occurs during the simulations.21 The RMSD, Rg and eccentricity of Cyt-c on Au(111) are slightly larger when compared with those of other systems. This is mainly due to the partial structural change in the Ω1 loop upon adsorption, as has been discussed above. Interestingly, the RMSD value of Cyt-c on COOH-SAM is even lower than in bulk solution, indicating the adsorption of Cyt-c on this surface helps to preserve its native structure. The dipole moment of Cyt-c in bulk solution (251 D) is approximate to that of its crystal structure (255 D). However, it is slightly larger for Cyt-c

20


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adsorbed on negtively- charged COOH-SAM (313 D) than that of Cyt-c in other systems. This is consistent with the previous simulation results18,

20-21

that the negatively-charged

COOH-SAM is inclined to increase the dipole moment of the adsorbed Cyt-c. Table 2. Averaged properties of Cyt-c adsorbed on surfaces and in bulk solution. Heme System

Dipole

Orientation

tilt angle

Rg

angle

(Degree)

(Å)

Crystal

-

-

12.64

Bulk

-

-

Au(111)

−0.18±0.11

COOH-SAM

−0.91±0.05

Eccentricity

RMSD

moment

(Å)

(Debye)

0.144

-

255

12.87

0.149

1.13

251±29

129±4

13.02

0.156

1.55

284±34

74±8

12.87

0.146

0.91

313±36

3.6. Mechanism for the Hindrance of Electron Transfer The hindrance of ET of Cyt-c adsorbed on bare gold surface has been disputably ascribed to the formation of aggregates12, unfolding or even denaturation of the protein14, 16, or the orientation changes of the heme15, 17, as has been described in the introduction. However, the simulation results in this work and the recent experiments have proved that the secondary structure of Cyt-c is well preserved when adsorbed onto the bare gold surface. It has been proposed that the ET of the adsorbed Cyt-c is determined by the interplay between protein dynamics and tunneling probabilities.21 For the protein dynamics, it exerts two level of tuning on the electronic coupling through reorientation (coarse) and low amplitude thermal fluctuations (fine).21 In order to make electron transfer, Cyt-c undergoes a rearrangement of the heme with respect to the electrode surface by altering its conformation or orientation.21, 40 For example, the deactivation of redox properties of Cyt-c on a highly charged surface has been ascribed to the hindrance of the 21



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repositioning movement of the heme caused by the strong electrostatic interactions between Cyt-c and the negatively charged surface.40 Our simulation results have shown that Cyt-c has a narrower distribution of heme tilt angle, in other words, Cyt-c has a more concentrated orientation distribution on the bare gold surface than on the COOH-SAM surface. Therefore, the hindrance of ET for Cyt-c adsorbed on bare gold surface is caused by the confined rotation of the heme group. For the tunneling probabilities, a large number of studies have shown that the nonadiabatic (tunneling) ET mechanism58 predicts the exponential decay of the ET rate constant with the ET distance Re. The ET rate constant ket is subjected to the following relationship:

ket ∝ exp  − β ( Re − Ro ) 

(2)

where Ro is a minimum distance of the electron donor-acceptor and β is a decay parameter which relies on the intervening atomic and molecular structure.58 Further calculations show that the distance of the central iron of heme to the Au (111) surface (about 12.9 Å) is relatively farther than that to the COOH-SAM surface (about 10.6 Å, which is in accordance with previous simulations21). This is another reason for the hindrance of ET of Cyt-c adsorbed on bare gold surface. 4. CONCLUSIONS In this work, the adsorption orientation and conformation of Cyt-c on Au(111) surface were investigated by performing PTMC and MD simulations. The results reveal that the most favorable binding mode of Cyt-c on the gold surface agrees with the results characterized by SEIRA spectroscopy. The adsorption is mainly contributed by the strong vdW interactions between residues that have long side chains and the gold surface, which leads to the helix A 22


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and Ω1 loop being in contact with the surface and most of the α-helixes running nearly parallel to the gold surface. The native structure of Cyt-c is well preserved during the adsorption and only the flexible Ω1 loop and the N-terminal show a relatively larger mobility. Cyt-c has a more concentrated distribution of heme tilt angle on the bare gold surface than that on COOH-SAM surface. Furthermore, the distance of the central iron of heme to the Au (111) surface (about 12.9 Å) is relatively farther than that to the COOH-SAM surface (about 10.6 Å). This may be the reason for the hindrance of ET for Cyt-c on bare gold surface.

ASSOCIATED CONTENT

Supporting Information Details on the evolution of the interaction energies between Cyt-c and the Au(111) surface (Figure S1), the evolution of the heme tilt angle (Figure S2) and dipole direction of Cyt-c (Figure S3) with respect to the surface normal during the MD simulations. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS This work is funded by the National Key Basic Research Program of China (No. 2013CB733500), National Natural Science Foundation of China (Nos. 21376089, 91334202), Guangdong Science Foundation (No. 2014A030312007) and State Key Laboratory of Materials-Oriented Chemical Engineering (KL12-05). The computational resources for this project are provided by SCUTGrid at South China University of Technology.

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Table of contents only

31



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Figure 1. Secondary structure assignment of horse heart Cyt-c and illustration of the orientation angles. Cytc is displayed in NewCartoon mode and the heme is represented in Licorice mode. The three arrows: blue (n), black (m) and red (e) show the direction of surface normal, heme plane normal and the dipole, respectively. 71x71mm (300 x 300 DPI)


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Figure 2. Orientation distributions of the dipole of Cyt-c on different surfaces by PTMC simulations. The blue, red and black lines correspond to the positively charged, negatively charged and neutral surfaces, respectively. 70x49mm (300 x 300 DPI)



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Figure 3. Initial and final configurations for MD simulations of Cyt-c adsorbed on Au(111) surface. (a)-(c) are the three preferred orientations from PTMC simulations; (d) and (e) are from experimental results; (f)-(j) are the corresponding configurations after MD simulations starting from (a)-(e). The red arrows indicate the direction of the dipole. Water and ions along with the dipole atoms and virtual sites of Au(111) surface are not shown for clarity. 79x35mm (300 x 300 DPI)


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Figure 4. The most favorable orientations of Cyt-c adsorbed on Au(111) and COOH-SAM surfaces. The upper row and the lower row are for the side-view and top-view configurations, respectively. Residues less than 3.5 Å from the surface are represented in Licorice mode and colored by ResType. For top view, only the portion of Cyt-c near the surface is shown and the surfaces are shown in transparent mode. 89x97mm (300 x 300 DPI)



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Figure 5. Interaction energies between contact residues of Cyt-c and the (a) Au(111) and (b) COOH-SAM surfaces 62x22mm (300 x 300 DPI)


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Figure 6. Orientation distributions of Cyt-c adsorbed on Au(111) and COOH-SAM surfaces by MD simulations for (a) the dipole angle, and (b) heme tilt angle. 80x109mm (300 x 300 DPI)



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Figure 7. The mobility (RMSF) of backbone atoms per residue of Cyt c in bulk solution and on Au(111) and COOH-SAM surfaces. Inset: simulated structure of Cyt-c in bulk solution (cyan) and on Au(111) (yellow) and COOH-SAM (red) surfaces superimposed on its crystal structure (blue). 58x41mm (300 x 300 DPI)


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TOC graphic 49x52mm (300 x 300 DPI)


Molecular Simulations of Cytochrome c Adsorption on a Bare Gold

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