A Hydrogen Bond Between Linear Tetrapyrrole and Conserved Aspartate Causes the Far-Red Shifted Absorption of Phytochrome Photoreceptors

Photoswitching of phytochrome photoreceptors between red-absorbing (Pr) and far-red absorbing (Pfr) states triggers light adaptation of plants, bacteria and other organisms. Using quantum chemistry, we elucidate the color-tuning mechanism of phytochromes and identify the origin of the Pfr-state red-shifted spectrum. Spectral variations are explained by resonance interactions of the protonated linear tetrapyrrole chromophore. In particular, hydrogen bonding of pyrrole ring D with the strictly conserved aspartate shifts the positive charge towards ring D thereby inducing the red spectral shift. Our MD simulations demonstrate that formation of the ring D–aspartate hydrogen bond depends on interactions between the chromophore binding domain (CBD) and phytochrome specific domain (PHY). Our study guides rational engineering of fluorescent phytochromes with a far-red shifted spectrum

Insight into the structural dynamics of light sensitive proteins from time-resolved crystallography and quantum chemical calculations

International audienceThe structural dynamics underlying molecular mechanisms of light-sensitive proteins can be studied by a variety of experimental and computational biophysical techniques. Here we review recent progress in combining time-resolved crystallography at X-ray free electron lasers and quantum chemical calculations to study structural changes in photoenzymes, photosynthetic proteins, photoreceptors, and photoswitchable fluorescent proteins following photoexcitation

Four Resonance Structures Elucidate Double-Bond Isomerisation of a Biological Chromophore

Photoinduced double-bond isomerisation of the chromophore of photoactive yellow protein (PYP) is highly sensitive to chromophore-protein interactions. On the basis of high-level ab initio calculations, using the XMCQDPT2 method, we scrutinise the effect of the chromophore-protein hydrogen bonds on the photophysical and photochemical properties of the chromophore. We identify four resonance structures – two closed-shell and two biradicaloid – that elucidate the electronic structure of the ground and first excited states involved in the isomerisation process. Changing the relative energies of the resonance structures by hydrogen-bonding interactions tunes all photochemical properties of the chromophore in an interdependent manner. Our study sheds new light on the role of the chromophore electronic structure in tuning in photosensors and fluorescent proteins

Role of the Molecular Environment in Flavoprotein Color and Redox Tuning: QM Cluster versus QM/MM Modeling

We investigate the origin of the excitation energy shifts induced by the apoprotein in the active site of the bacterial photoreceptor BLUF (<u>B</u>lue <u>L</u>ight sensor <u>U</u>sing <u>F</u>lavin adenine dinucleotide). In order to compute the vertical excitation energies of three low-lying electronic states, including two π–π* states of flavin (S₁ and S₂) and a π–π* tyrosine-flavin electron-transfer state (ET), with respect to the energy of the closed-shell ground state (S₀), we prepared alternative quantum mechanical (QM) cluster and quantum mechanics/molecular mechanics (QM/MM) models. We found that the excitation energies computed with both types of models correlate with the magnitude of the charge transfer character of the excitation. Accordingly, we conclude that the small charge transfer character of the light absorbing S₀–S₁ transition and the substantial charge transfer character of the nonabsorbing but redox active S₀–ET transition explain the small color changes but substantial redox tuning in BLUF and also in other flavoproteins. Further analysis showed that redox tuning is governed by the electrostatic interaction in the QM/MM model and transfer of charge between the active site and its environment in the QM cluster. Moreover, the wave function polarization of the QM subsystem by the MM subsystem influences the magnitude of the charge transfer, resulting in the QM/MM and QM excitation energies that are not entirely consistent

Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic Reappraisal

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX. Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state

Protonation States of Molecular Groups in the Chromophore-Binding Site Modulate Properties of the Reversibly Switchable Fluorescent Protein rsEGFP2

The role of protonation states of the chromophore and its neighboring amino acid side chains of the reversibly switching fluorescent protein rsEGFP2 upon photoswitching is characterized by molecular modeling methods. Numerous conformations of the chromophore-binding site in computationally derived model systems are obtained using the quantum chemistry and QM/MM approaches. Excitation energies are computed using the extended multiconfigurational quasidegenerate perturbation theory (XMCQDPT2). The obtained structures and absorption spectra allow us to provide interpretation of the observed structural and spectral properties of rsEGFP2 in the active ON- and inactive OFF-states. To identify intermediates along the routes of chromophore transformations between the ON- and OFF-states, molecular dynamics trajectories with the QM/MM potentials are examined. The results demonstrate that in addition to the dominating anionic and neutral forms of the chromophore, the cationic and zwitterionic forms may participate in the photoswitching of rsEGFP2. Conformations and protonation forms of the Glu223 and His149 side chains in the chromophore-binding site play an essential role in stabilizing specific protonation forms of the chromophore

Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry

Domratcheva T, Hartmann E, Schlichting I, Kottke T. Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry. Sci. Rep. 2016;6(1): 22669

Challenges in Computing Electron-Transfer Energies of DNA Repair Using Hybrid QM/MM Models

The influence of the molecular environment on chemical activity is an important factor in biomolecular mechanisms. We studied the effects of ionic groups, that is, a protonated histidine side chain and deprotonated phosphates of DNA, on electron transfer in light-induced DNA repair. On the basis of the X-ray crystal structure, we prepared a hybrid QM/MM model of the macromolecular complex formed between the (6–4) photolyase enzyme and the DNA substrate containing the thymine–thymine (6–4) photoproduct. At the optimized geometries, we computed with the CASSCF and CASPT2 methods the excited states of the electron donor and electron acceptor complex, consisting of the reduced flavin and the (6–4) photoproduct. The donor–acceptor complex interacts with its environment comprised of the protein, the double-stranded DNA substrate with its counterions, and the solvating water molecules, which we modeled using the AMBER94 force field. The excited states of our interest include two locally excited (LE) states of the flavin chromophore and intermolecular electron-transfer (ET) states. We identify only minor changes of the LE excitation energies by interactions with the environment, but in drastic contrast to that, we found significant changes of the ET excitation energies. In the presence of the positively charged His365H⁺, the ET excitation energies decrease, indicating facilitated electron transfer. In addition, the excitation energy of the second LE state, explaining the flavin’s absorption at 380 nm, undergoes a 0.2 eV downshift. In contrast to the active-site protonation, reduced screening of the DNA phosphates increases the ET excitation energies but not the LE excitation energies. Accordingly, the electron affinities of the (6–4) photoproduct are significantly reduced, which should hinder electron transfer from the excited flavin. We also show that dynamic electron correlation accounted by the second order perturbation theory CASPT2 does not alter the energy trends obtained with the CASSCF method. Including the histidine side chain in the QM part enhances the effect of the histidine protonation on the ET energies. We also note that protonated His365H⁺ can serve as an electron acceptor

Quantum-based modeling of protein-ligand interaction: The complex of RutA with uracil and molecular oxygen

Modern quantum-based methods are employed to model interaction of the flavin-dependent enzyme RutA with the uracil and oxygen molecules. This complex presents the structure of reactants for the chain of chemical reactions of monooxygenation in the enzyme active site, which is important in drug metabolism. In this case, application of quantum-based approaches is an essential issue, unlike conventional modeling of protein-ligand interaction with force fields using molecular mechanics and classical molecular dynamics methods. We focus on two difficult problems to characterize the structure of reactants in the RutA-FMN-O2-uracil complex, where FMN stands for the flavin mononucleotide species. First, location of a small O2 molecule in the triplet spin state in the protein cavities is required. Second, positions of both ligands, O2 and uracil, must be specified in the active site with a comparable accuracy. We show that the methods of molecular dynamics with the interaction potentials of quantum mechanics/molecular mechanics theory (QM/MM MD) allow us to characterize this complex and, in addition, to surmise possible reaction mechanism of uracil oxygenation by RutA