A Single-Point Mutation in d-Arginine Dehydrogenase Unlocks a Transient Conformational State Resulting in Altered Cofactor Reactivity.

Proteins are inherently dynamic, and proper enzyme function relies on conformational flexibility. In this study, we demonstrated how an active site residue changes an enzyme's reactivity by modulating fluctuations between conformational states. Replacement of tyrosine 249 (Y249) with phenylalanine in the active site of the flavin-dependent d-arginine dehydrogenase yielded an enzyme with both an active yellow FAD (Y249F-y) and an inactive chemically modified green FAD, identified as 6-OH-FAD (Y249F-g) through various spectroscopic techniques. Structural investigation of Y249F-g and Y249F-y variants by comparison to the wild-type enzyme showed no differences in the overall protein structure and fold. A closer observation of the active site of the Y249F-y enzyme revealed an alternative conformation for some active site residues and the flavin cofactor. Molecular dynamics simulations probed the alternate conformations observed in the Y249F-y enzyme structure and showed that the enzyme variant with FAD samples a metastable conformational state, not available to the wild-type enzyme. Hybrid quantum/molecular mechanical calculations identified differences in flavin electronics between the wild type and the alternate conformation of the Y249F-y enzyme. The computational studies further indicated that the alternate conformation in the Y249F-y enzyme is responsible for the higher spin density at the C6 atom of flavin, which is consistent with the formation of 6-OH-FAD in the variant enzyme. The observations in this study are consistent with an alternate conformational space that results in fine-tuning the microenvironment around a versatile cofactor playing a critical role in enzyme function.

A new interpretation of the absorption and the dual fluorescence of Prodan in solution.

Remarkable interest is associated with the interpretation of the Prodan fluorescent spectrum. A sequential hybrid Quantum Mechanics/Molecular Mechanics method was used to establish that the fluorescent emission occurs from two different excited states, resulting in a broad asymmetric emission spectrum. The absorption spectra in several solvents were measured and calculated using different theoretical models presenting excellent agreement. All theoretical models [semiempirical, time dependent density functional theory and and second-order multiconfigurational perturbation theory] agree that the first observed band at the absorption spectrum in solution is composed of three electronic excitations very close in energy. Then, the electronic excitation around 340 nm-360 nm may populate the first three excited states (π-π*Lb, n-π*, and π-π*La). The ground state S0 and the first three excited states were analyzed using multi-configurational calculations. The corresponding equilibrium geometries are all planar in vacuum. Considering the solvent effects in the electronic structure of the solute and in the solvent relaxation around the solute, it was identified that these three excited states can change the relative order depending on the solvent polarity, and following the minimum path energy, internal conversions may occur. A consistent explanation of the experimental data is obtained with the conclusive interpretation that the two bands observed in the fluorescent spectrum of Prodan, in several solvents, are due to the emission from two independent states. Our results indicate that these are the n-π* S2 state with a small dipole moment at a lower emission energy and the π-π*Lb S1 state with large dipole moment at a higher emission energy.

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming.

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly 10-fold increase in red-light emission, the second is not emissive. Thus, the W76S/Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multiconfigurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

Fluorescence Properties of Flavin Semiquinone Radicals in Nitronate Monooxygenase.

Fluorescent cofactors like flavins can be exploited to probe their local environment with spatial and temporal resolution. Although the fluorescence properties of the oxidized and two-electron-reduced states of flavins have been studied extensively, this is not the case for the one-electron-reduced state. Both the neutral and anionic semiquinones have proven particularly challenging to examine, as they are unstable in solution and are transient, short-lived species in many catalytic cycles. Here, we report that the nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 is capable of stabilizing both semiquinone forms anaerobically for hours, thus enabling us to study their spectroscopy in a constant protein environment. We found that in the active site of NMO, the anionic semiquinone exhibits no fluorescence, whereas the neutral semiquinone radical shows a relatively strong fluorescence, with a behavior that violates the Kasha-Vavilov rule. These fluorescence properties are discussed in the context of time-dependent density functional theory calculations, which reveal low-lying dark states in both systems.

Electronic spectra of flavin in different redox and protonation states: a computational perspective on the effect of the electrostatic environment.

Flavins are versatile molecules due to their ability to exist in multiple redox and protonation states. At physiological conditions, they are usually encountered either as oxidized quinones, neutral semiquinones, anionic semiquinones, neutral hydroquinones, or anionic hydroquinones. We compute the electronic near-UV/vis spectra for flavin in each of these five states. Specifically, we compute vertical, adiabatic, and vibronic excitations for all excited states that have wavelengths longer than 300 nm. We employ the calculations to assign the peaks in the corresponding experimental UV/vis spectra from literature. We also compare the effect of polar and non-polar solvents on the spectra using a polarizable continuum model. Finally, we construct "electrostatic spectral tuning maps" for prominent peaks in each of the five states. These maps qualitatively describe how the flavin electronic spectra will be shifted by an anisotropic electrostatic environment such as a protein. Understanding how flavin's UV/vis absorption spectrum is modulated by its environment can aid in experiments employing flavin as a probe of internal electrostatics of a protein and in engineering new color variants of flavoproteins.

Solvent Effects on the Dynamic Polarizability and Raman Response of Molecule-Metal Oxide Hybrid Clusters.

Currently, there is considerable interest in the properties of semiconducting metal oxide nanoparticle substrates because of their utility in surface-enhanced Raman scattering, dye-sensitized solar cells, and photocatalysis. While the enhancement of Raman activities of molecules adsorbed on these nanoparticles is due to a large increase in the polarizability, because of charge transfer from the molecule to the semiconducting nanoparticle, little is known about the factors responsible for modulating the polarizability, particularly the influence of the solvent. Consequently, we have carried out Monte Carlo simulations of several hybrids to study the solvent effect on the dynamic polarizabilities and electronic spectra. Our results indicate that the presence of the solvent induces a shift and an increase in the polarization response that is dependent on the identity of the hybrid. The observed enhancement can be attributed to both the resonant character of the excitation and the participation of the solvent in the charge redistribution. The methodology employed in this work could be very valuable in both identifying and developing metal oxides as novel molecular sensors.

Solvent effect on the Stokes shift and on the nonfluorescent decay of the daidzein molecular system.

The flavonoids have been the target of several experimental works due to its influence in the human health as antioxidant elements. The fluorescence properties of these compounds have been widely studied due to the large Stokes shifts experimentally observed and the variety of processes that lead to the fluorescence. In the present work the role of the solvent in the large Stokes shift experimentally observed in the daidzein molecular system in water is theoretically studied. Also studied is the nonfluorescent decay mechanism in a polar aprotic solvent like acetonitrile. The solvent effect in the ground and in the low-lying excited electronic states is taken into account by using the sequential-QM/MM methodology. Excited state properties like equilibrium geometries and transition energies were studied by using multiconfigurational calculations, CASSCF and CASPT2. The excited electronic state responsible for the fluorescence spectrum in water was identified, and the large Stokes shift seems to be the result of the large interaction of the system in this electronic state with the solvent. On the other hand, spin-orbit coupling calculations, between the singlet and triplet electronic states, indicate favorable conditions for intersystem crossing, in agreement with the experimental result of nonfluorescence observation.

Alternative Strategy for Spectral Tuning of Flavin-Binding Fluorescent Proteins.

iLOV is an engineered flavin-binding fluorescent protein (FbFP) with applications for in vivo cellular imaging. To expand the range of applications of FbFPs for multicolor imaging and FRET-based biosensing, it is desirable to understand how to modify their absorption and emission wavelengths (i.e., through spectral tuning). There is particular interest in developing FbFPs that absorb and emit light at longer wavelengths, which has proven challenging thus far. Existing spectral tuning strategies that do not involve chemical modification of the flavin cofactor have focused on placing positively charged amino acids near flavin's C4a and N5 atoms. Guided by previously reported electrostatic spectral tunning maps (ESTMs) of the flavin cofactor and by quantum mechanical/molecular mechanical (QM/MM) calculations reported in this work, we suggest an alternative strategy: placing a negatively charged amino acid near flavin's N1 atom. We predict that a single-point mutant, iLOV-Q430E, has a slightly red-shifted absorption and fluorescence maximum wavelength relative to iLOV. To validate our theoretical prediction, we experimentally expressed and purified iLOV-Q430E and measured its spectral properties. We found that the Q430E mutation results in a slight change in absorption and a 4-8 nm red shift in the fluorescence relative to iLOV, in good agreement with the computational predictions. Molecular dynamics simulations showed that the carboxylate side chain of the glutamate in iLOV-Q430E points away from the flavin cofactor, which leads to a future expectation that further red shifting may be achieved by bringing the side chain closer to the cofactor.

Theoretical study of the absorption and nonradiative deactivation of 1-nitronaphthalene in the low-lying singlet and triplet excited states including methanol and ethanol solvent effects.

The photophysics of the 1-nitronaphthalene molecular system, after the absorption transition to the first singlet excited state, is theoretically studied for investigating the ultrafast multiplicity change to the triplet manifold. The consecutive transient absorption spectra experimentally observed in this molecular system are also studied. To identify the electronic states involved in the nonradiative decay, the minimum energy path of the first singlet excited state is obtained using the complete active space self-consistent field//configurational second-order perturbation approach. A near degeneracy region was found between the first singlet and the second triplet excited states with large spin-orbit coupling between them. The intersystem crossing rate was also evaluated. To support the proposed deactivation model the transient absorption spectra observed in the experiments were also considered. For this, computer simulations using sequential quantum mechanic-molecular mechanic methodology was used to consider the solvent effect in the ground and excited states for proper comparison with the experimental results. The absorption transitions from the second triplet excited state in the relaxed geometry permit to describe the transient absorption band experimentally observed around 200 fs after the absorption transition. This indicates that the T(2) electronic state is populated through the intersystem crossing presented here. The two transient absorption bands experimentally observed between 2 and 45 ps after the absorption transition are described here as the T(1)→T(3) and T(1)→T(5) transitions, supporting that the intermediate triplet state (T(2)) decays by internal conversion to T(1).

CASPT2 study of the potential energy surface of the HSO2 system.

The importance of the HSO(2) system in atmospheric and combustion chemistry has motivated several works dedicated to the study of associated structures and chemical reactions. Nevertheless controversy still exists in connection with the reaction SH + O(2)→ H + SO(2) and also related to the role of the HSOO isomers in the potential energy surface (PES). Here we report high-level ab initio calculation for the electronic ground state of the HSO(2) system. Energetic, geometric, and frequency properties for the major stationary states of the PES are reported at the same level of calculations: CASPT2/aug-cc-pV(T+d)Z. This study introduces three new stationary points (two saddle points and one minimum). These structures allow the connection of the skewed HSOO(s) and the HSO(2) minima defining new reaction paths for SH + O(2) → H + SO(2) and SH + O(2) → OH + SO. In addition, the location of the HSOO isomers in the reaction pathways have been clarified.

The effect of hydrogen-bonding on flavin's infrared absorption spectrum.

Cluster and continuum solvation computational models are employed to model the effect of hydrogen bonding interactions on the vibrational modes of lumiflavin. Calculated spectra were compared to experimental Fourier-transform infrared (FTIR) spectra in the diagnostic 1450-1800 cm-1 range, where intense νC=C, νC=N, [Formula: see text] , and [Formula: see text] stretching modes of flavin's isoalloxazine ring are found. Local mode analysis is used to describe the strength of hydrogen-bonding in cluster models. The computations indicate that νC=C and νC=N mode frequencies are relatively insensitive to intermolecular interactions while the [Formula: see text] and [Formula: see text] modes are sensitive to direct (and also indirect for [Formula: see text] ) hydrogen-bonding interactions. Although flavin is neutral, basis sets without the diffuse functions provide incorrect relative frequencies and intensities. The 6-31+G* basis set is found to be adequate for this system, and there is limited benefit to considering larger basis sets. Calculated vibrational mode frequencies agree with experimentally determined frequencies in solution when cluster models with multiple water molecules are used. Accurate simulation of relative FTIR band intensities, on the other hand, requires a continuum (or possibly quantum mechanical/molecular mechanical) model that accounts for long-range electrostatic effects. Finally, an experimental peak at ca. 1624 cm-1 that is typically assigned to the [Formula: see text] vibrational stretching mode has a complicated shape that suggests multiple underlying contributions. Our calculations show that this band has contributions from both the C6-C7 and C2 = O stretching vibrations.

Ionic Atmosphere Effect on the Absorption Spectrum of a Flavoprotein: A Reminder to Consider Solution Ions.

This study utilizes the FMN-dependent NADH:quinone oxidoreductase from Pseudomonas aeruginosa PAO1 to investigate the effect of introducing an active site negative charge on the flavin absorption spectrum both in the absence and presence of a long-range electrostatic potential coming from solution ions. There were no observed changes in the flavin UV-visible spectrum when an active site tyrosine (Y277) becomes deprotonated in vitro. These results could only be reproduced computationally using average solvent electrostatic configuration (ASEC) QM/MM simulations that include both positive and negative solution ions. The same calculations performed with minimal ions to neutralize the total protein charge predicted that deprotonating Y277 would significantly alter the flavin absorption spectrum. Analyzing the distribution of solution ions indicated that the ions reorganize around the protein surface upon Y277 deprotonation to cancel the effect of the tyrosinate on the flavin absorption spectrum. Additional biochemical experiments were performed to test this hypothesis.

Free Energy Computation for an Isomerizing Chromophore in a Molecular Cavity via the Average Solvent Electrostatic Configuration Model: Applications in Rhodopsin and Rhodopsin-Mimicking Systems.

We present a novel technique for computing the free energy differences between two chromophore "isomers" hosted in a molecular environment (a generalized solvent). Such an environment may range from a relatively rigid protein cavity to a flexible solvent environment. The technique is characterized by the application of the previously reported "average electrostatic solvent configuration" method, and it is based on the idea of using the free energy perturbation theory along with a chromophore annihilation procedure in thermodynamic cycle calculations. The method is benchmarked by computing the ground-state room-temperature relative stabilities between (i) the cis and trans isomers of prototypal animal and microbial rhodopsins and (ii) the analogue isomers of a rhodopsin-like light-driven molecular switch in methanol. Furthermore, we show that the same technology can be used to estimate the activation free energy for the thermal isomerization of systems i-ii by replacing one isomer with a transition state. The results show that the computed relative stability and isomerization barrier magnitudes for the selected systems are in line with the available experimental observation in spite of their widely diverse complexity.

Computational and Spectroscopic Characterization of the Photocycle of an Artificial Rhodopsin.

The photocycle of a reversible photoisomerizing rhodopsin mimic (M2) is investigated. This system, based on the cellular retinoic acid binding protein, is structurally different from natural rhodopsin systems, but exhibits a similar isomerization upon light irradiation. More specifically, M2 displays a 15-cis to all-trans conversion of retinal protonated Schiff base (rPSB) and all-trans to 15-cis isomerization of unprotonated Schiff base (rUSB). Here we use hybrid quantum mechanics/molecular mechanics (QM/MM) tools coupled with transient absorption and cryokinetic UV-vis spectroscopies to investigate these isomerization processes. The results suggest that primary rPSB photoisomerization of M2 occurs around the C13═C14 double bond within 2 ps following an aborted-bicycle pedal (ABP) isomerization mechanism similar to natural microbial rhodopsins. The rUSB isomerization is much slower and occurs within 48 ps around the C15═N double bond. Our findings reveal the possibility to engineer naturally occurring mechanistic features into artificial rhodopsins and also constitute a step toward understanding the photoisomerization of UV pigments. We conclude by reinforcing the idea that the presence of the retinal chromophore inside a tight protein cavity is not mandatory to exhibit ABP mechanism.

Electrostatic Spectral Tuning Maps for Biological Chromophores.

The mechanism by which the absorption wavelength of a molecule is modified by a protein is known as spectral tuning. Spectral tuning is often achieved by electrostatic interactions that stabilize/destabilize or modify the shape of the excited and ground-state potential energy surfaces of the chromophore. We present a protocol for the construction of three-dimensional "electrostatic spectral tuning maps" that describe how vertical excitation energies in a chromophore are influenced by nearby charges. The maps are built by moving a charge on the van der Waals surface of the chromophore and calculating the change in its excitation energy. The maps are useful guides for protein engineering of color variants, for interpreting spectra of chromophores that act as probes of their environment, and as starting points for further quantum mechanical/molecular mechanical studies. The maps are semiquantitative and can approximate the magnitude of the spectral shift induced by a point charge at a given position with respect to the chromophore. We generate and discuss electrostatic spectral tuning maps for model chromophores of photoreceptor proteins, fluorescent proteins, and aromatic amino acids. Such maps may be extended to other properties such as oscillator strengths, absolute energies (stability), ionization energies, and electron affinities.

Impact of Electronic State Mixing on the Photoisomerization Time Scale of the Retinal Chromophore.

Spectral data show that the photoisomerization of retinal protonated Schiff base (rPSB) chromophores occurs on a 100 fs time scale or less in vertebrate rhodopsins, it is several times slower in microbial rhodopsins and it is between one and 2 orders of magnitude slower in solution. These time scale variations have been attributed to specific modifications of the topography of the first excited state potential energy surface of the chromophore. However, it is presently not clear which specific environment effects (e.g., electrostatic, electronic, or steric) are responsible for changing the surface topography. Here, we use QM/MM models and excited state trajectory computations to provide evidence for an increase in electronic mixing between the first and the second excited state of the chromophore when going from vertebrate rhodopsin to the solution environments. Ultimately, we argue that a correlation between the lifetime of the first excited state and electronic mixing between such state and its higher neighbor, may have been exploited to evolve rhodopsins toward faster isomerization and, possibly, light-sensitivity.

An Average Solvent Electrostatic Configuration Protocol for QM/MM Free Energy Optimization: Implementation and Application to Rhodopsin Systems.

A novel atomistic methodology to perform free energy geometry optimization of a retinal chromophore covalently bound to any rhodopsin-like protein cavity is presented and benchmarked by computing the absorption maxima wavelengths (λmax) of distant rhodopsin systems. The optimization is achieved by computing the Nagaoka's Free Energy Gradient (FEG) within an Average Solvent Electrostatic Configuration (ASEC) atomistic representation of the thermodynamic equilibrium and minimizing such quantity via an iterative procedure based on sequential classical MD and constrained QM/MM geometry optimization steps. The performance of such an ASEC-FEG protocol is assessed at the CASPT2//CASSCF/Amber level by reproducing the λmax values observed for 12 mutants of redesigned human cellular retinol binding protein II (hCRBPII) systems; a set of 10 distant wild-type rhodopsins from vertebrates, invertebrates, eubacteria, and archaea organisms; and finally a set of 10 rhodopsin mutants from an eubacterial rhodopsin. The results clearly show that the proposed protocol, which can be easily extended to any protein incorporating a covalently bound ligand, yields correct λmax trends with limited absolute errors.

Toward Automatic Rhodopsin Modeling as a Tool for High-Throughput Computational Photobiology.

We report on a prototype protocol for the automatic and fast construction of congruous sets of QM/MM models of rhodopsin-like photoreceptors and of their mutants. In the present implementation the information required for the construction of each model is essentially a crystallographic structure or a comparative model complemented with information on the protonation state of ionizable side chains and distributions of external counterions. Starting with such information, a model formed by a fixed environment system, a flexible cavity system, and a chromophore system is automatically generated. The results of the predicted vertical excitation energy for 27 different rhodopsins including vertebrate, invertebrate, and microbial pigments indicate that such basic models could be employed for predicting trends in spectral changes and/or correlate the spectral changes with structural variations in large sets of proteins.