Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence—A Theoretical Study

Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light‐emitting products, and 4a‐hydroxy‐5‐hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high‐level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron‐transfer mechanism.     

Understanding the complete bioluminescence cycle from a multiscale computational perspective: A review

Bioluminescence (BL) is an amazing natural phenomenon whose visible light is produced by living organisms. BL phenomenon is quite pervasive and has been observed in 17 phyla of 4 kingdoms. This fascinating natural phenomenon has unceasingly attracted people’s curiosity from ancient era to today. For a very long time, we can only receive some sporadic and static information from experimental observations, the mechanism of most BL remains is unclear. How the chemical reaction of BL process is initiated? Where the energy for light emission comes from? How does the light emitter produce? What is the light emitter for a wild bioluminescent organism? How to regain luciferin for next bioluminescence when it is used up? The luciferin is utilized forthwith or stored and release for subsequent light emission? What factors affect the color and strength of a bioluminescence? How to artificially tune the bioluminescence for special application? Computational BL plays unreplaceable role in answering these mechanistic questions. In contrast with experimental BL, computational BL came very late. In the past two decades, computational BL has touched nearly all the bioluminescent systems with chemical bases via the method of multiscale simulation. In this review, the author firstly introduced the history, types and general chemical process of BL. Then, the computational scheme on BL was briefly epitomized. Using firefly BL as a paradigmatic case, the author summarized theoretical investigation on the six stages of general chemical process in a BL cycle: luciferin oxidation, peroxide thermolysis, light emission, luciferin regeneration, luciferin storage and luciferin release. At each stage, the available theoretical studies of other bioluminescent organisms are briefly introduced and compared with the firefly system. Basing on the mechanistic understanding, the author reviewed the up-to-date theoretical design on bioluminescent systems. Again, the firefly was mainly focused on, and the other possible systems were just briefly introduced. This review summarized the theoretical studies to date on BL and addressed the status, critical challenges and future prospects of computational BL.     

Mechanism of proteolysis in matrix metalloproteinase‐2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase‐2 (MMP‐2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all‐atom three‐dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace‐Gln‐Gly～Ile‐Ala‐Gly‐Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four‐step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and C? N bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate‐limiting step in MMP‐2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations. © 2015 Wiley Periodicals, Inc.     

All‐Optical Integrated Logic Operations Based on Chemical Communication between Molecular Switches

Molecular logic gates process physical or chemical “inputs” to generate “outputs” based on a set of logical operators. We report the design and operation of a chemical ensemble in solution that behaves as integrated AND, OR, and XNOR gates with optical input and output signals. The ensemble is composed of a reversible merocyanine‐type photoacid and a ruthenium polypyridine complex that functions as a pH‐controlled three‐state luminescent switch. The light‐triggered release of protons from the photoacid is used to control the state of the transition‐metal complex. Therefore, the two molecular switching devices communicate with one another through the exchange of ionic signals. By means of such a double (optical–chemical–optical) signal‐transduction mechanism, inputs of violet light modulate a luminescence output in the red/far‐red region of the visible spectrum. Nondestructive reading is guaranteed because the green light used for excitation in the photoluminescence experiments does not affect the state of the gate. The reset is thermally driven and, thus, does not involve the addition of chemicals and accumulation of byproducts. Owing to its reversibility and stability, this molecular device can afford many cycles of digital operation.     

Modeling emission features of salicylidene aniline molecular crystals: A QM/QM’ approach

A new computational protocol relying on the use of electrostatic embedding, derived from QM/QM’ ONIOM calculations, to simulate the effect of the crystalline environment on the emission spectra of molecular crystals is here applied to the β‐form of salicylidene aniline (SA). The first singlet excited states (S₁) of the SA cis‐keto and trans‐keto conformers, surrounded by a cluster of other molecules representing the crystalline structure, were optimized by using a QM/QM’ ONIOM approach with and without electronic embedding. The model system consisting of the central salicylidene aniline molecule was treated at the DFT level by using either the B3LYP, PBE0, or the CAM‐B3LYP functional, whereas the real system was treated at the HF level. The CAM‐B3LYP/HF level of theory provides emission energies in good agreement with experiment with differences of ?20/?32 nm ( cis‐keto form) and ?8/?14 nm ( trans‐keto form), respectively, whereas notably larger differences are obtained using global hybrids. Though such differences on the optical properties arise from the density functional choice, the contribution of the electronic embedding is rather independent of the functional used. This plays in favor of a more general applicability of the present protocol to other crystalline molecular systems. © 2016 Wiley Periodicals, Inc.     

Interfacing the GROMOS (bio)molecular simulation software to quantum‐chemical program packages

The newly implemented quantum‐chemical/molecular‐mechanical (QM/MM) functionality of the Groningen molecular simulation (GROMOS) software for (bio)molecular simulation is described. The implementation scheme is based on direct coupling of the GROMOS C++ software to executables of the quantum‐chemical program packages MNDO and TURBOMOLE, allowing for an independent further development of these packages. The new functions are validated for different test systems using program and model testing techniques. The effect of truncating the QM/MM electrostatic interactions at various QM/MM cutoff radii is discussed and the application of semiempirical versus density‐functional Hamiltonians for a solute molecule in aqueous solution is compared. © 2012 Wiley Periodicals, Inc.     

Theoretical Study of Dinoflagellate Bioluminescence

Dinoflagellates are the most ubiquitous luminescent protists in the marine environment and have drawn much attention for their crucial roles in marine ecosystems. Dinoflagellate bioluminescence has been applied in underwater target detection. The luminescent system of dinoflagellates is a typical luciferin–luciferase one. However, the excited‐state oxyluciferin is not the light emitter of dinoflagellate bioluminescence as in most luciferin–luciferase bioluminescent organisms. The oxyluciferin of bioluminescent dinoflagellates is not fluorescent, whereas its luciferin emits bright fluorescence with similar wavelength of the bioluminescence. What is the light emitter of dinoflagellate bioluminescence and what is the chemical process of the light emission like? These questions have not been answered by the limited experimental evidence so far. In this study, for the first time, the density functional calculation is employed to investigate the geometries and properties of luciferin and oxyluciferin of bioluminescent dinoflagellate. The calculated results agree with the experimental observations and indicate the luciferin or its analogue, rather than oxyluciferin, is the bioluminophore of dinoflagellate bioluminescence. A rough mechanism involving energy transfer is proposed for dinoflagellate bioluminescence.     

Investigation of the Hydroxylation Mechanism of Noncoupled Copper Oxygenases by Ab Initio Molecular Dynamics Simulations

In Nature, the family of copper monooxygenases comprised of peptidylglycine α‐hydroxylating monooxygenase (PHM), dopamine β‐monooxygenase (DβM), and tyramine β‐monooxygenase (TβM) is known to perform dioxygen‐dependent hydroxylation of aliphatic C? H bonds by using two uncoupled metal sites. In spite of many investigations, including biochemical, chemical, and computational, details of the C? H bond oxygenation mechanism remain elusive. Herein we report an investigation of the mechanism of hydroxylation by PHM by using hybrid quantum/classical potentials (i.e., QM/MM). Although previous investigations using hybrid QM/MM techniques were restricted to geometry optimizations, we have carried out ab initio molecular dynamics simulations in order to include the intrinsic flexibility of the active sites in the modeling protocol. The major finding of this study is an extremely fast rebound step after the initial hydrogen‐abstraction step promoted by the cupric–superoxide adduct. The hydrogen‐abstraction/rebound sequence leads to the formation of an alkyl hydroperoxide intermediate. Long‐range electron transfer from the remote copper site subsequently triggers its reduction to the hydroxylated substrate. We finally show two reactivity consequences inherent in the new mechanistic proposal, the investigation of which would provide a means to check its validity by experimental means.     

A conclusive mechanism of the photoinduced reaction cascade in blue light using flavin photoreceptors

On the basis of extensive first-principle calculations within the framework of quantum mechanics/molecular mechanics (QM/MM), a conclusive mechanism for the formation of the signaling state of blue light using flavin (BLUF) domain proteins is proposed which is compatible with the experimental data presently available. Time-dependent density functional, as well as advanced coupled cluster response theory was employed for the QM part in order to describe the relevant excited states. One of the key residues involved in the mechanism is the glutamine adjacent to the flavin chromophore. The reaction cascade, triggered by the initial photoexcitation of the flavin chromophore, involves isomerization of this residue but no rotation as assumed previously. The fact that only the environment, but not the flavin chromophore by itself, is chemically transformed along the individual steps of the mechanism is unique for biological photoreceptors. The final isomer of the glutamine tautomer, i.e., the imidic acid, is further stabilized by the interchange of a methionine residue in the binding pocket with a tryptophan residue. The flip of these two residues might be the trigger for the large conformational change of this protein which is consequently transmitted as the signal to the biological environment.     

QM/MM Investigations on the Bioluminescent Decomposition of Coelenterazine Dioxetanone in Obelin

The bioluminescent mechanism of colenterazine dioxetanone(CZD) in the photoprotein of Obelia(obelin) was investigated by the combined quantum and molecular mechanics(QM/MM) method at TD-DFT level, which involved the real protein environment in decomposition of 1,2-dioxetanones. The anionic decomposition of CZD in (CZD+H₂O)^- model can go through a charge transfer(CT) catalyzed asynchronous-concerted process, which can be elucidated by the gradual reversible CT initiated luminescence(GRCTIL) mechanism. The neutral CZD in (CZDH+H₂O) decomposes through an uncatalyzed non-CT biradical process. The anionic decomposition catalyzed by CT, in which the S₀/S₁ surface "double crossing" hence has ability to provide high quantum yield of singlet chemiexcitation is thus more possible in bioluminescence of photoprotein.     

Revisiting the Origin of Bacterial Bioluminescence: QM/MM Study on Oxygenation Reaction of Reduced Flavin in Protein

Bacterial bioluminescence is initiated by the oxygenation reaction of reduced flavin mononucleotide in luciferase. This enzymatic oxygenation occurs in a wide range of biological processes including cellular redox metabolism, biocatalysis, biosynthesis and homeostasis. However, little is known about the mechanism of the enzymatic reaction between singlet reduced flavin and triplet oxygen. To explore the enigmatic oxygenation, for the first time, the reaction of reduced flavin anion with oxygen was studied in bacterial luciferase by a combined quantum mechanics and molecular mechanics method as well as molecular dynamics simulation. The calculated results demonstrate that the reaction proceeds via a proton-coupled electron transfer (PCET) pathway, and the essential αHis44 acts as a catalytic acid to provide the proton. The currently proposed PCET mechanism clearly describes the initial steps of bacterial bioluminescence, and could be suitable for the other flavin oxygenation reactions in enzymes.     

On the effect of a variation of the force field, spatial boundary condition and size of the QM region in QM/MM MD simulations

During the past years, the use of combined quantum-classical, QM/MM, methods for the study of complex biomolecular processes, such as enzymatic reactions and photocycles, has increased considerably. The quality of the results obtained from QM/MM calculations is largely dependent on five aspects to be considered when setting up a molecular model: the QM Hamiltonian, the MM Hamiltonian or force field, the boundary and coupling between the QM and MM regions, the size of the QM region and the boundary condition for the MM region. In this study, we systematically investigate the influence of a variation of the molecular mechanics force field and the size of the QM region in QM/MM MD simulations on properties of the photoactive part of the blue light photoreceptor protein AppA. For comparison, we additionally performed classical MD simulations and studied the effect of a variation of the type of spatial boundary condition. The classical boundary conditions and the force field used in a QM/MM MD simulation are shown to have non-neglegible effects upon the structural and energetic properties of the protein which makes it advisable to minimize computational artifacts in QM/MM MD simulations by application of periodic boundary conditions and a thermodynamically calibrated force field. A comparison of the structural and energetic properties of MD simulations starting from two alternative, different X-ray structures for the blue light utilizing flavin protein in its dark state indicates a slight preference of the two force fields used for the so-called Anderson structure over the Jung structure.     

Dynamic structures of phosphodiesterase-5 active site by combined molecular dynamics simulations and hybrid quantum mechanical/molecular mechanical calculations

Various quantum mechanical/molecular mechanical (QM/MM) geometry optimizations starting from an x-ray crystal structure and from the snapshot structures of constrained molecular dynamics (MD) simulations have been performed to characterize two dynamically stable active site structures of phosphodiesterase-5 (PDE5) in solution. The only difference between the two PDE5 structures exists in the catalytic, second bridging ligand (BL2) which is HO- or H2O. It has been shown that, whereas BL2 (i.e. HO-) in the PDE5(BL2 = HO-) structure can really bridge the two positively charged metal ions (Zn2+ and Mg2+), BL2 (i.e. H2O) in the PDE5(BL2 = H2O) structure can only coordinate Mg2+. It has been demonstrated that the results of the QM/MM geometry optimizations are remarkably affected by the solvent water molecules, the dynamics of the protein environment, and the electronic embedding charges of the MM region in the QM part of the QMM/MM calculation. The PDE5(BL2 = H2O) geometries optimized by using the QM/MM method in different ways show strong couplings between these important factors. It is interesting to note that the PDE5(BL2 = HO-) and PDE5(BL2 = H2O) geometries determined by the QM/MM calculations neglecting these three factors are all consistent with the corresponding geometries determined by the QM/MM calculations that account for all of these three factors. These results suggest the overall effects of these three important factors on the optimized geometries can roughly cancel out. However, the QM/MM calculations that only account for some of these factors could lead to considerably different geometries. These results might be useful also in guiding future QM/MM geometry optimizations on other enzymes.     

Computational characterization of reaction intermediates in the photocycle of the sensory domain of the AppA blue light photoreceptor

The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark-adapted and light-induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical-molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc-pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS-CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo-excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediates.     

Theoretical Insights into Chirality‐Controlled SWCNT Growth from a Cycloparaphenylene Template

A self‐assembly mechanism for low‐temperature SWCNT growth from a [6]cycloparaphenylene ([6]CPP) precursor via ethynyl (C₂H) radical addition is presented, based on non‐equilibrium quantum chemical molecular dynamics (QM/MD) simulations and density functional theory (DFT) calculations. This mechanism, which maintains the (6,6) armchair chirality of a SWCNT fragment throughout the growth process, is energetically more favorable than a previously proposed Diels–Alder‐based growth mechanisms [E. H. Fort, et al., J. Mater. Chem. 2011 , 21, 1373]. QM/MD simulations and DFT calculations show that C₂H radicals play dual roles during SWCNT growth, by abstracting hydrogen from the SWCNT fragment and providing the carbon source necessary for growth itself. Simulations demonstrate that chirality‐controlled SWCNT growth from macrocyclic hydrocarbon seed molecules with pre‐selected edge structure can be accomplished when the reaction conditions are carefully selected for hydrogen abstraction by radical species during the growth process.     

Solving Blue Light Riddles: New Lessons from Flavin‐binding LOV Photoreceptors

Detection of blue light (BL) via flavin‐binding photoreceptors (Fl‐Blues) has evolved throughout all three domains of life. Although the main BL players, that is light, oxygen and voltage (LOV), blue light sensing using flavins (BLUF) and Cry (cryptochrome) proteins, have been characterized in great detail with respect to structure and function, still several unresolved issues at different levels of complexity remain and novel unexpected findings were reported. Here, we review the most prevailing riddles of LOV‐based photoreceptors, for example: the relevance of water and/or small metabolites for the dynamics of the photocycle; molecular details of light‐to‐signal transduction events; the interplay of BL sensing by LOV domains with other environmental stimuli, such as BL plus oxygen‐mediating photodamage and its impact on microbial lifestyles; the importance of the cell or chromophore redox state in determining the fate of BL‐driven reactions; the evolutionary pathways of LOV‐based BL sensing and associated functions through the diverse phyla. We will discuss major novelties emerged during the last few years on these intriguing aspects of LOV proteins by presenting paradigmatic examples from prokaryotic photosensors that exhibit the largest complexity and richness in associated functions.     

Quantum Mechanics/Molecular Mechanics Study on the Photoreactions of Dark‐ and Light‐Adapted States of a Blue‐Light YtvA LOV Photoreceptor

The dark‐ and light‐adapted states of YtvA LOV domains exhibit distinct excited‐state behavior. We have employed high‐level QM(MS‐CASPT2)/MM calculations to study the photochemical reactions of the dark‐ and light‐adapted states. The photoreaction from the dark‐adapted state starts with an S₁→T₁ intersystem crossing followed by a triplet‐state hydrogen transfer from the thiol to the flavin moiety that produces a diradical intermediate, and a subsequent internal conversion that triggers a barrierless C−S bond formation in the S₀ state. The energy profiles for these transformations are different for the four conformers of the dark‐adapted state considered. The photochemistry of the light‐adapted state does not involve the triplet state: photoexcitation to the S₁ state triggers C−S bond cleavage followed by recombination in the S₀ state; both these processes are essentially barrierless and thus ultrafast. The present work offers new mechanistic insights into the photoresponse of flavin‐containing blue‐light photoreceptors.     

量子力学和分子力学组合方法及其应用

QM/MM组合方法在研究凝聚态中的化学反应及生物大分子的结构和活性之间的关系等方面已取得重要进展。这一方法的要点在于将大体系配分成几部分,根据需要对不同部分进行不同级别的处理,因此既利用了量子力学的精确性,又利用了分子力学的高效性。对QM/MM组合理论及其一些最新进展作一简单介绍,并以最近进行了几个工作为例说明QM、MM组合方法的应用。     

One‐Pot Bioconversion of l‐Arabinose to l‐Ribulose in an Enzymatic Cascade

This work reports the one‐pot enzymatic cascade that completely converts l ‐arabinose to l ‐ribulose using four reactions catalyzed by pyranose 2‐oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild‐type P2O is specific for the oxidation of six‐carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l ‐arabinose at the C2‐position. The variant T169G was identified as the best candidate, and this had an approximately 40‐fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild‐type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4‐OH of l ‐arabinose, which allows a reduction in the distance between the C2‐H and flavin N5, facilitating hydride transfer and enabling flavin reduction.     

The Origin and Dynamics of Soft X‐Ray‐Excited Optical Luminescence of ZnO

The distinct optical emission from ZnO materials, nanoneedles and microcrystallites synthesized with different sizes and morphologies by a flow deposition technique, is investigated with X‐ray excited optical luminescence (XEOL) and time‐resolved X‐ray excited optical luminescence (TR‐XEOL) from a synchrotron light source at the O K and Zn L_3,2 edges. The innovative use of XEOL, allowing site‐specific chemical information and luminescence information at the same time, is fundamental to provide direct evidence for the different behaviour and the crucial role of bulk and surface defects in the origin of ZnO optical emission, including dynamics. XEOL from highly crystalline ZnO nanoneedles is characterized by a sharp band‐gap emission (～380 nm) and a broad red luminescence (～680 nm) related to surface defects. Luminescence from ZnO microcrystallites is mostly dominated by green emission (～510 nm) associated with defects in the core. TR‐XEOL experiments show considerably faster decay dynamics in nanoneedles compared to microcrystallites for both band‐gap emission and visible luminescence. Herein we make a fundamental step forward correlating for the first time the interplay of size, crystallinity, morphology and excitation energy with luminescence from ZnO materials.