Importance of the Intramolecular Hydrogen Bond on the

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Importance of the Intramolecular Hydrogen Bond on the Photochemistry of Anionic Hydroquinone (FADH-) in DNA Photolyase Yue-Jie Ai,†,‡ Feng Zhang,†,‡ Shu-Feng Chen,† Yi Luo,*,‡,§ and Wei-Hai Fang*,† †

College of Chemistry, Beijing Normal University, Beijing 100875, China, ‡Department of Theoretical Chemistry, School of Biotechnology, Royal Institute of Technology, S-106 91 Stockholm, Sweden, and §Hefei National Laboratory for Physical Sciences at the Microscale, Department of Chemical Physics, University of Science and Technology of China, Hefei, Anhui 230026, China

ABSTRACT The design of a proper molecular model with a good balance between the size of the model system and the computational capacity is essential for theoretical modeling of biological systems. We have shown in this Letter that the often used model system, a lumiflavin (7,8-dimethy-10-methylisoalloxazine), cannot correctly describe geometrical and electronic structures of FADH- in DNA photolyase. The intramolecular hydrogen bond between the isoalloxazine ring and the ribityl moiety is found to play a significant role in controlling photochemical properties of FADH- in DNA photolyase. SECTION Biophysical Chemistry

D

NA photolyases are flavoproteins that are responsible for the repair photocycle of UV-damaged DNA. The key cofactor in DNA photolyase is flavin molecules, which can exist in three different oxidation states, the oxidized form (flavoquinone), radical form (flavosemiquinone), and reduced form (flavohydroquinone). It is believed that the catalytic state in vivo is the fully reduced form of anionic hydroquinone (FADH-) in photolyase,1 as shown in Figure 1. With the help of an electron-transfer redox cycle of the FADHmolecule, the photolyase breaks down the mutagenic photoproduct cyclobutane pyrimidine dimers (CPDs) by absorbing energy from UV light to repair damaged DNA.2 In recent years, because of the important functionality of FADH- in the DNA repairing process, the photophysics and photochemistry of flavins in DNA photolyase have attracted considerable attention in both experimental2-4 and theoretical5,6 studies. The most widely used theoretical model for the FADHmolecule is a lumiflavin (7,8-dimethy-10-methylisoalloxazine),5-8 which is the active core of FADH-, as indicated in Figure 1. However, by closely inspecting the structure of the FADH- molecule taken from Escherichia coli,9 one can see that there is a hydrogen bond formed between the 30 -OH group of the ribityl moiety and the N1 atom of the isoalloxazine ring; see Figure 1. From a pure structure point of view, such an intramolecular hydrogen bond can stabilize the lumiflavin core and hinder the motion of the isoalloxazine ring, which in turn could affect the dynamics of photochemical processes in DNA photolyases. Very recently, Kao et al.4 have studied excited-state dynamics of FADH- and other flavin molecules at different oxidation states in both solution and protein environments with femtosecond laser spectroscopy. It has been found that the dynamic behavior of the studied system in the solution is very

r 2010 American Chemical Society

different from that in the protein, which was attributed to the ring flexibility of the flavin.4 In light of the new experimental evidence, the role of the intramolecular hydrogen bond on the electronic structure and photochemistry of the FADH- molecule in the solution and the protein becomes an important issue, which has been completely overlooked in previous theoretical studies. The goal of this Letter is to reveal the effect of the intramolecular hydrogen bond on the electronic structure and the optical absorption/emisson of the FADH- molecule in solution and protein. Our calculations have indicated that such an intramolecular hydrogen bond should always be included in theoretical models for studying DNA photolyses. In our study, two models of FADH-, with and without the intramolecular hydrogen bond, have been chosen for comparison. The conventional model M-NHD (molecule with no intramolecular hydrogen bond) has only the isoalloxazine ring, as shown in Figure 2a, while the new model M-IHD (molecule with intramolecular hydrogen bond) in Figure 3a includes the hydroxyl alkyl to take into account the intramolecular hydrogen bond. All initial coordinates were taken from the crystal structure of the DNA photolyase enzyme from E. coli9 (PDB code: 1DNP) with manually added hydrogens. The geometries of these two models were optimized at the CASSCF(10e,8o)/6-31G(d) level. For the small model, a larger basis set 6-31G(d,p) was also used, which gave almost the same structures. For the case of the solution, specific water molecules were included in both models, as shown in

Received Date: December 20, 2009 Accepted Date: January 26, 2010 Published on Web Date: January 29, 2010

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DOI: 10.1021/jz900434z |J. Phys. Chem. Lett. 2010, 1, 743–747

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protein, for M-NHD, there were two degenerate states, the socalled convex and concave conformations15 in the ground states. The butterfly bending angle is found to be about 26°. However, when the intramolecular hydrogen bond is included, that is, the M-IHD model, the energy profile of the ground state is completely changed. The degeneracy of the ground state in M-NHD is removed, leading to two minima. The convex bending structure S01 is about 5 kcal/mol lower in energy than another minimum S02, which has a very flat ring structure. The bending angles of S01 and S02 are predicted to be 14 and 9°, respectively. It is interesting to note that in the experimental crystal structure of Mees,16 the reduced form of flavin was found to have a bending angle of approximately 9°, which is very close to our result for M-IHD. The intramolecular hydrogen bond between the 30 -OH and N1 in these two minima are 1.950 and 1.914 Å, respectively. This intramolecular hydrogen bond in FADH- is much stronger than that in the neutral radical state of FADH, which was reported to be 2.38 Å.17 In solution, it can be expected that the water molecule breaks down this intramolecular hydrogen bond in M-IHD to form an intermolecular hydrogen bond with the 30 OH group and the N1 atom of the isoalloxazine ring. This process results in only one minimum with a bending angle of 13° in the ground state. Our calculations clearly demonstrate that the inclusion of the intramolecular hydrogen bond can have significant impact on the geometry of the system. The M-IHD model is more consistent with the experimental findings than the widely used M-NHD model. It was proposed in the previous experimental study4 that the motion of the isoalloxazine ring is mainly restricted by the environment around the cofactor, while in the solution

Figures 2d and 3d, respectively. Single-point TDDFT calculations for the ground and excited states were carried out in combination with a polarizable continuum model (IEFPCM)10-12 in the equilibrium (eq) time regime. Static dielectric constants of 4 and 78.39 were adopted to mimic the protein and water environments, respectively. The linear coupling model (LCM)13 was used to compute absorption and emission spectra; see the Supporting Information for details. All calculations were performed with the Gaussian 03 package.14 It has been under debate for many years whether oxidation states of flavins should be of a planar or a “butterfly” conformation. The current consensus seems to be that the oxidized and radical species have a planar conformation while the reduced one (like FADH-) has a folded butterfly conformation at its equilibrium. The degree of the butterfly bending is described by the dihedral angle C10aN5N10C9a here. In the

Figure 1. Structure and labeling of the flavin in its fully reduced form, FADH-. The active core isoalloxazine is shown within the dashed lines.

Figure 2. Geometries (a) and (c) and the electronic structure (b) of two degenerate ground states for the M-NHD model for FADH- in the photolyase. Geometry (d) with five water molecules and the electronic structures (e) for M-NHD for FADH- in the solution.


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conventional M-NHD is not capable of explaining the experimental findings. A more realistic model with inclusion of the intramolecular hydrogen bond is necessary for describing the geometric and electronic states of FADH-. In the protein environment, the intramolecular hydrogen bond in M-IHD limits the freedom of the isoalloxazine ring of M-NHD and results in two minima, while in the solution, this intramolecular hydrogen bond in M-IHD is replaced by an intermolecular hydrogen bond that bridges a water molecule and FADH-. It seems like that the restriction of the flexibility of the isoalloxazine ring in the photolyase actually originates mainly from the molecule itself, that is, the intramolecular hydrogen bond between the 30 -OH group of the ribityl moiety and the N1 atom of the isoalloxazine ring in the protein environment. The replacement of such an intramolecular hydrogen bond with an intermolecular hydrogen bond in the solution results in different photodynamics. One way to further highlight the importance of the intramolecular hydrogen bond is to directly calculate emission spectra and to compare with experiments. We have adopted the linear coupling model for all systems under investigation. With the LCM, the emission profile is a mirror image of the absorption. For large system like M-IHD, we could not obtain Hessian matrices of all minima with CASSCF. It is simply beyond the capacity of the program packages that we have used.

environment, such a restriction no longer exists and the flavin become much more flexible. Because of this, the rotation or distortion of the isoalloxazine ring caused by the butterfly bending motion in the solution facilitates the excited-state dynamics of flavins to the time scale of picoseconds.4 Such a hypothesis can be tested by calculating the deactivation pathway of the FADH- molecule in solution as a function of the ring rotation. Our TDDFT calculations have found that the rotation of the isoalloxazine ring in M-NHD along the N5-N10 axis has negligible influence on electronic states of the molecule. In other words, the flexibility of the isoalloxazine ring itself can not be the main deactivation factor. Moreover, for M-NHD, we have located the conical intersection (CI) of the S0 and the S1 states, which is about 1.49 eV above the equilibrium of the S1 state. One could thus conclude that the CI might not be the dominate decay path that leads to the ultrafast deactivation dynamics as anticipated.4 More computational details can be found in the Supporting Information. Unfortunately, we are not able to carry out a full energy potential search for the M-IHD model with the intramolecular hydrogen bond at a reasonable CAS level using the computational codes to which we have access. It would be very interesting to see how the presence of the intramolecular hydrogen bond changes the conical intersection of the S0 and the S1 states. Our calculations have at least shown that the

Figure 3. Geometries (a) and (c) and the electronic structure (b) of two ground states for the M-IHD model for FADH- in the photolyase. Geometry (d) with five water molecules and the electronic structures (e) for FADH- in the solution.


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Figure 4. Calculated emission spectra of M-NHD and M-IHD models in the protein and in the solution environment.

led to significant changes in the emission spectra but has also brought the theoretical results closer to the experiments. In summary, we have examined the effects of the intramolecular hydrogen bond on geometric and electronic structures of model systems for FADH- in DNA photolyase by ab initio calculations. It is found that the intramolecular hydrogen bond between the 30 -OH group of the ribityl moiety and the N1 atom of the isoalloxazine ring in the photolyase is very important for good descriptions of chemical and photochemical properties of FADH- in DNA photolyase. It is our suggestion that such an intramolecular hydrogen bond should always be included in model systems.

Instead, we used Hessian matrices obtained at the B3LYP level with the 6-31G(d) basis set to carry out the frequency analysis. For a small model of M-NHD, calculations show that the vibrational normal modes obtained at the CAS and B3LYP levels are very similar. The computed emission spectra for the M-NHD and M-IHD models are shown in Figure 4. The maxima of the spectral profiles of M-NHD and M-IHD in the protein are calibrated with respect to the experimental maximum in the protein, while the results for other systems are shifted accordingly by the same amount. In other words, the relative energy changes of the calculated spectra in the protein and in the solution are kept. It can be clearly seen for both M-NHD and M-IHD models that their emission spectral profiles are very different in the protein and in the solution. The presence of an intermolecular hydrogen bond with water molecules results in the blue shift of the spectrum and the narrowing of the spectral width. The effect of the intramolecular hydrogen bond on the emission spectral profile is also very strong. In general, it induces similar changes as the intermolecular hydrogen bond, namely, narrowing of the spectral width. This is due to the fact that the inclusion of the intramolecular hydrogen bond restricts the motion of the molecule and makes many vibrational modes inactive. The consequence of it is to cut off the long tail at the low energy (longer wavelength) of the spectrum of M-NHD (see Figure 4) in the protein and to make the double peaks at the higher energy (short wavelength) resolvable. The reduction of the spectral width has, on the other hand, simplified the emission of the molecule in the solution; the emission spectrum of M-IHD in the solution shows only one narrow peak. It is interesting to see that the calculated spectra for M-IHD in the protein and in the solution are in very reasonable agreement with the experimental ones4 with respect to the general spectral shapes. Moreover, it was found experimentally4 that the spectrum in solution shows a blue shift of 0.45 eV with respect to the maximum of the spectrum in the protein, while our calculations give a value (0.30 eV) quite close to the experiments. Moreover, the calculated double peaks of M-IHD are found to be 510 and 545 nm in the protein, in a good agreement with the experimental values of 515 and 545 nm, respectively.4 From a spectroscopic point view, the inclusion of the intramolecular hydrogen bond has not only


SUPPORTING INFORMATION AVAILABLE LCM theory discussion, equilibrium structures, potential energy surface, and the root mean square deviation of the vibrational frequency. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author: *To whom correspondence should be addressed. E-mail: luo@ kth.se (Y.L.); [email protected] (W.-H.F.).

ACKNOWLEDGMENT The work was supported by the Swedish

Research Council (VR), the Swedish National Infrastructure for Computing (SNIC), the NSFC (20720102038, 20925311), and the Major State Basic Research Development Programs (2004CB719903, 2010CB923300).

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