DFT modeling of water-assisted hydrogen peroxide formation from a C(4a)-(hydro)peroxyflavin.

The cofactor of a class A monooxygenase is reduced at an external location of the enzyme and is subsequently pulled back into the active site after the reduction. This observation brings the question; is there any defense mechanism of the active site of a monooxygenase against the formation of the harmful hydrogen peroxide from the reactive C(4a)-(hydro)peroxide intermediate? In this study, the barrier energies of one to three water molecule-mediated uncoupling reaction mechanisms in water exposed reaction conditions were determined. These were found to be facile barriers. Secondly, uncoupling was modeled in the active site of kynurenine 3-monooxygenase complex which was represented with 258 atoms utilizing cluster approach. Comparison of the barrier energy of the cluster model to the models that represent the water exposed conditions revealed that the enzyme does not have an inhibitory reaction site architecture as the compared barrier energies are roughly the same. The main defense mechanism of KMO against the formation of the hydrogen peroxide is deduced to be the insulation, and without this insulation, the monooxygenation would not take place as the barrier height of the hydrogen peroxide formation within the active site is almost half of that of the monooxygenation.

Modelling the elusive conformational activation in kynurenine 3-monooxygenase.

Kynurenine 3-monooxygenase (KMO) regulates the levels of important physiological intermediates in the kynurenine pathway [Guillemin, et al., Journal of Neuroscience, 2007, 27, 12884], which is the major route for L-tryptophan catabolism. Its catalytic activity (hydroxylation) is dependent on the formation of a short-lived intermediate that forms after the reduction of the coenzyme FAD. The reduction takes place fast when the substrate binds to KMO. Crystal structures of the apo form and in complex with an effector inhibitor, which prevents the hydroxylation of the substrate but also stimulates KMO like the substrate, and a competitive inhibitor, which suppresses the substrate hydroxylation, are available for the resting in conformation only. The active out conformational state that enables the reduction of FAD at an exposed location of KMO after its stimulation by an effector, however, was implicated but not resolved experimentally and has remained elusive so far. Molecular dynamics simulations of apo KMO and the inhibitor-KMO complexes are carried out using extensive multi-dimensional umbrella sampling to explore the free-energy surface of the coenzyme FAD's conformational conversion from the in state (buried within the active site) to the out state. This allows a discussion and comparison with the experimental results, which showed a significant increase in the rate of reduction of FAD in the presence of an effector inhibitor and absence of enzymatic function in the presence of a competitive inhibitor [Kim, et al., Cell Chemical Biology, 2018, 25, 426]. The free-energy barriers associated with those conformational changes and structural models for the active out conformation are obtained. The interactions during the conformational changes are determined to identify the influence of the effector.

A methionine biomolecule-modified chromenylium-cyanine fluorescent probe for the analysis of Hg2+ in the environment and living cells.

The objective of the study is, for the first time, to construct a new near infrared (NIR) fluorophore, spectrophotometric, colorimetric, ratiometric, and turn-on probe (CSME) based on chromenylium cyanine platform decorated with methionine biomolecule to provide an efficient solution for critical shortcoming to be encountered for analysis of hazardous Hg2+ in environment and living cell. The CSME structure and its interaction with Hg2+ ion were evaluated by NMR, FTIR, MS, UV-Vis and fluorescence methods as well as Density Functional Theory (DFT) calculations. The none fluorescence CSME having spirolactam ring only interacted with Hg2+ in aqueous solution including competing ions. This interaction caused the fluorescence CSME with opened spirolactam form which exhibited spectral and colorimetric changes in the NIR region. The probe based on UV-Vis and fluorescence techniques respond in 90 s, has wide linear ranges (for UV-Vis: 6.29 × 10-8 - 1.86 × 10-4 M; for fluorescence: 9.49 × 10-9 - 1.13 × 10-5 M), and has a lower Limit of Detection (LOD) value (for fluorescence: 4.93 × 10-9 M, 0.99 ng/mL) than the value predicted by the US Environmental Protection Agency (EPA) organization. Hg2+ analysis was performed in drinking and tap water with low Relative Standard Deviation (RSD) values and high recovery. Smartphone and living cell applications were successfully performed for colorimetric sensing Hg2+ in real samples and 3T3 cells, respectively.

A new highly Selective, sensitive and NIR spectrophotometric probe based on A2B2-Type of unsymmetrical phthalocyanine for hazardous Be2+ recognition.

The aim of this work is to construct a new A2B2-type of unsymmetrical and ratiometric phthalocyanine (Pc) based-probe O-A2B2ZnPc to provide an effective solution to critical inadequacy to be experienced for the detection of hazardous Be2+. O-A2B2ZnPc enabling strong absorption and emission in Near-Infrared region (λabs-λem wavelengths of 694-712 nm) showed excellent selectivity and sensitivity toward Be2+ among competitive metal ions by both spectrophotometric and fluorometric methods. The probe with oligomeric Pc form in THF was degraded with the addition of aqueous Be2+ and arranged to J-aggregation form, resulting in a remarkably diminishing in Q-band at 694 nm as well as a new band formation at 746 nm, and a considerably decreasing in fluorescence emission. The probe has superior features for the determination of Be2+ such as high quantum efficiency and photochemical stability, rapid response (1 s), high selectivity and very low Limit of Detection (0.26 ppb and 1.5 ppb) for UV-Vis and fluorescence, respectively which are quite good values according to the permissible amount of Be2+ (4 ppb) in water as specified by World Health Organization. O-A2B2ZnPc can be shown among the best performing probes with its unique properties according to previous studies in the literature. In addition, the geometrical and spectral features of the O-A2B2ZnPc were analyzed in detail by DFT calculations.

Computational Survey of Recent Experimental Developments in the Hydroxylation Mechanism of Kynurenine 3-Monooxygenase.

Recently, two new mechanistic proposals for the kynurenine 3-monooxygenase (KMO) catalyzed hydroxylation reaction of l-Kynurenine (l-Kyn) have been proposed. According to the first proposal, instead of the distal oxygen, the proximal oxygen of the hydroperoxide intermediate of flavin adenine dinucleotide (FAD) is transferred to the substrate ring. The second study proposes that l-Kyn participates in its base form in the reaction. To address these proposals, the reaction was reconsidered with a 386 atom quantum cluster model that is based on a recent X-ray structure (PDB id: 6FOX). The computations were carried out at the UB3LYP/6-311+G(2d,2p)//UB3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections. To supplement the results of the density functional theory (DFT) calculations, molecular dynamics (MD) simulations of the protein-substrate complex were employed. The comparison of a proximal oxygen transfer mechanism to the distal oxygen transfer mechanism revealed that the former requires too high of a barrier energy while the latter validated our previous results. According to the MD simulations, the hydroperoxy moiety does not favor an alignment that might promote the proximal oxygen transfer mechanism. In the second part of the study, hydroxylation reaction with the base form of l-Kyn was sought. Although DFT calculations confirmed a much more facile reaction with the base form of l-Kyn, a mechanism which would allow the deprotonation of the l-Kyn before the oxygen transfer could not be determined with the X-ray-based positions. A concerted mechanism with both the oxygen transfer and the deprotonation required a high barrier energy. A stepwise mechanism involving the deprotonation of l-Kyn was found, starting from an MD frame. The overall barrier of the oxygen transfer step of this model was found to be in the range of that of with neutral l-Kyn. MD simulations supported the idea of ineffectiveness of the nearby shell surrounding the utilized active site core on the deprotonation of l-Kyn.

In silico methods predict new blood-brain barrier permeable structure for the inhibition of kynurenine 3-monooxygenase.

Kynurenine 3-monooxygenase (KMO) regulates the levels of bioactive substances in the kynurenine pathway of tryptophan catabolism and its activity is tied to so many diseases that finding an appropriate inhibitor for KMO has become an urgent task. This especially proved to be difficult for the central nervous system related diseases due to the requirement that the supposed inhibitor should be both blood brain barrier permeable and should not cause hydrogen peroxide as a harmful side product. In this in silico study, we present our step-wise approach, whose starting point is based on the important experimental observations. To tackle the problem, a library of 7561938 structures was obtained from Zinc15 database utilizing the tranche browser. From this library, a subset of 501777 structures was determined with the considerations of their functional groups that constrain their applicability. Then, the binding affinity ranking of this set of structures was determined via virtual screening. Starting from the structures whose affinities are the highest among this subset, the ADMET properties were checked through in silico methods and the binding properties of the selected inhibitor candidates were further investigated via molecular dynamics simulations and MM/GBSA calculations. According to the computational results of this study, ZINC_71915355 has passed all the evaluations and is a potentially BBB permeable structure that can inhibit KMO. Additionally, ZINC_19827377 was identified as a new potential KMO inhibitor which may be more suitable for peripheral administration. From the in silico study presented herein, ZINC_71915355 and ZINC_19827377 structures, which showed high binding affinity without harmful H2O2 production, along with the tailored properties can now serve as powerful candidates for KMO inhibition and these hits are worth of further experimental validation.

Mechanism of Kynurenine 3-Monooxygenase-Catalyzed Hydroxylation Reaction: A Quantum Cluster Approach.

The mechanism of the hydroxylation reaction between l-Kyn and model flavin adenine dinucleotide (FAD)-hydroperoxide was investigated via density functional theory (DFT) calculations in the absence and in the presence of the kynurenine 3-monooxygenase (KMO) enzyme by considering possible pathways that can lead to the product 3-hydroxykynurenine (3-HK). Crystal structure (pdb code: 5NAK )-based calculations involved a quantum cluster model in which the active site of the enzyme with the substrate l-Kyn was represented with 348 atoms. According to the deduced mechanism, KMO-catalyzed hydroxylation reaction takes place with four transformations. In the initial transition state, FAD delivers its peroxy hydroxyl to the l-Kyn ring, creating an sp3-hybridized carbon center. Then, the hydrogen on the hydroxyl moiety is immediately transferred back to the proximal oxygen that remained on FAD. These consequent transformations are in line with the somersault rearrangement previously described for similar enzymatic systems. The second step corresponds to a hydride shift from the sp3-hybridized carbon of the substrate ring to its adjacent carbon, producing the keto form of 3-HK. Then, keto-3-HK is transformed into its enol form (3-HK) with a water-assisted tautomerization. Lastly, FAD is oxidized with a water-assisted dehydration, which also involves 3-HK as a catalyst. In the proposed pathway, Asn54, Pro318, and a crystal water molecule were seen to play significant roles in the proton relays. The energies obtained via the cluster approach were calculated at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections.

Superior Sensor for Be2+ Ion Recognition via the Unprecedented Octahedral Crystal Structure of a One-Dimensional Coordination Polymer of Crown Fused Zinc Phthalocyanine.

The unprecedented one-dimensional (1-D) coordination polymer of crown fused zinc phthalocyanine (P-CfZnPc) with an octahedral crystal structure and with intermolecular packing that has superior multichannel sensor ability for Be2+ ion recognition was prepared and characterized by single-crystal X-ray diffraction analysis (XRD) and a wide range of spectroscopic and voltammetric methods. An exceptional feature of the crystal structure of P-CfZnPc is that each zinc ion in the phthalocyanine (Pc) polymer is coordinated by the four isoindole nitrogen atoms and an outer oxygen atom of the Pc molecule. This structure is the first example of an octahedral arrangement in a 1-D polymeric chain for zinc phthalocyanines (ZnPcs) and zinc porphyrins (ZnPs) reached without the presence of a coordinating solvent, which was confirmed by XRD analysis. Interestingly, this (1-D) coordination polymer preserves its conformation in THF (tetrahydrofuran) solution, thereby effectively preventing aggregation. This result was confirmed by the particle size of the molecule (125 nm) using dynamic light scattering (DLS) and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra as well as UV-vis spectroscopy. The sensor has long-term stability (more than 3 months in solution), a very low response time (less than 1 s), and nonaggregating ability, facilitating the accurate determination of ultra-trace amounts of Be2+ (lower than 1 ppb), which is extremely important in terms of human health and environmental protection. The sensor can highly selectively and sensitively bind Be2+ among Li+, Na+, K+, Cs+, Mg2+, Ca2+, Ba2+, Al3+, Co2+, Hg2+, Ni2+, Pb2+, and Zn2+ ions via Be2+-induced J aggregation of Pc molecules. Such a binding leads to not only a significant decrease in Pc absorption (677 nm) as well as the creation of new absorption (720 nm) but also fluorescence emission quenching (690 nm). Furthermore, the sensor displayed highly selective voltammetric recognition for Be2+ following J aggregation/disaggregation in the second reduction process. The binding mechanism of the sensor and Be2+ ion was also explained on the basis of TD-DFT calculations.