The dual nature of superoxide (O2•-) as both a damaging oxidant and a common intermediate in oxygen-dependent enzymatic reactions raises a fundamental question about how enzymes control its reactivity. Firefly luciferase (FLuc), which productively utilizes O2•- to drive its light-emitting reaction, provides an excellent model system to elucidate these control mechanisms. In this work, using a combination of quantum chemical, QM/MM, and QM/MM MD simulations, we demonstrate that this selectivity is governed by a preorganized active-site architecture. We show that a hydrogen-bonding network, primarily with the F246 backbone and active-site water molecules, stabilizes O2•-. At the same time, the G245-F246 backbone and F246 side chain sterically block unproductive additions of O2•- to the deprotonated luciferyl adenylate radical and orient O2•- for selective formation of the dioxetanone─the key intermediate of the light-emitting pathway. We also identify how the H244 side chain, protonated in the preceding reaction steps, can trigger a major unproductive side reaction producing the hydroperoxyl radical and subsequently hydrogen peroxide, and propose that this pathway may be potentially regulated by the pyrophosphate cofactor acting as a scavenger for excessive protons in the active site. Finally, we establish that the Mg2+ ion is essential for the final dioxetanone ring closure by charge neutralization, which reduces the electrostatic repulsion in the AMP cleavage step and prevents the formation of an adenylated dioxetanone analogue unproductive in light emission.

The high catalytic power of cholinesterases results from an efficient interplay between optimised structure, molecular dynamics and fine adjustment of substrates in the active site. Kinetic and molecular modelling investigations of the catalysis of 'inverse' ester substrates compared to 'normal' ester substrates by butyrylcholinesterase (BChE) were performed to shed light on the effect of isomeric inversion of the ester bond on binding and catalytic steps. The substrates used were phenyl-acetate (PhA), -propionate (PhPr), -butyrate (PhBu). The 'inverse' substrates used were benzoic acid methyl ester (BAME), phenyl acetic acid methyl ester (PAAME) and phenyl propionic acid methyl ester (PPAME). Inversion of the ester bond dramatically affected catalytic efficiency. Comparative analysis of the kinetics of both types of substrates showed that hydrolysis of 'inverse' substrates was only possible at high enzyme concentrations, and the rates were first-order, indicating (S)max << Km. The ratio of specific activity (kcat/Km) between normal and 'inverse' substrates increased 12-fold from PhA/BAME to PhPr/PAAME. PhBu was the best substrate, while PPAME was not hydrolysed by BChE. Thus, introduction of methylene group(s) in the acyl moiety of substrates progressively increased kcat/Km, while introduction of methylene group(s) in phenol/alcohol moiety of 'inverse' substrate considerably decreased kcat/Km, indicating dramatic loss of substrate complementarity to the active site. QM/MM and molecular dynamics simulations revealed the molecular basis for altered kinetics of 'inverse' substrate hydrolysis. The observed effects stemmed from reduced stability of reaction intermediates and transition states, along with inversion of the productive complex conformation. BChE exhibited no selectivity for this conformation or alternative binding modes.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein’s C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications.

The bioluminescence reaction of the firefly luciferase (FLuc) enzyme proceeds in two steps: protein-catalyzed formation of an active substrate from ATP and luciferin and subsequent interaction of the activated substrate with molecular oxygen resulting in emission of light. Here, we model the protein-dependent formation of the deprotonated luciferyl adenylate and uncover the catalytic role of the MgATP binding to the enzyme. Guided by the established structures of the ATP/GTP-binding proteins, we identify the MgATP binding loop in FLuc and model interactions of this motif with MgATP by means of classical and hybrid QM/MM molecular dynamics. Based on the free energy simulations, we demonstrate that MgATP bound to FLuc plays a dual mechanistic role: after ATP functioning as a substrate in luciferin adenylation, it catalyzes deprotonation of the luciferyl adenylate providing interactions that enhance the strength of the general base and stabilize the deprotonated intermediate. Moreover, the pyrophosphate PPi produced in the adenylation step is a plausible final proton acceptor enabling proton release from the active site. Our findings reveal a novel role of MgATP in generating a luciferin species primed for the reaction with molecular oxygen.

Cholinesterases (ChEs) are irreversibly inhibited by organophosphorus compounds (OP) through phosphylation of their active site serine. OPs can be regarded as pseudo-substrates of ChEs in which water-mediated dephosphylation is very slow or impossible. Then, it has been attempted by genetic engineering to convert ChEs into OP hydrolases by speeding up dephosphylation rates. The knowledge of ChEs crystal structure and implementation of potent in silico methods allow rational computer design of new mutants. Such ChE mutants could be used as catalytic bioscavengers in medical counter-measures of OP poisoning. In the present work, using QM/MM MD, we designed a new mutant of human butyrylcholinesterase (BChE). This double mutant, Glu325Gly/Asn322Glu, alters the functioning of the catalytic triad (Ser198.His438.Glu325 in wild-type enzyme), making a new one Ser198.His438.Glu322. Calculations with echothiophate as a model OP showed that the new catalytic triad is stable and capable of hydrolysing the phosphorylated serine. Thus, self-reactivation of the mutated enzyme is possible. Although the calculated dephosphylation rate constant is low, it is expected that the introduction of a third mutation will lead to a much faster turnover.

We report high-level electronic structure calculations of electronic states in the miniSOG (for mini Singlet Oxygen Generator) photoactive protein designed to produce singlet oxygen upon light exposure. We consider a model system with a riboflavin (RF) chromophore. To better understand the photosensitization process, we compute relevant electronic states of the combined oxygen-chromophore system and their couplings. The calculations suggest that singlet oxygen can be produced both by inter-system crossing, via a triplet state of the RF(T1)×O2(3Σ− g ) character as well as by triplet excitation energy transfer via a singlet state of the same character. The calculations also suggest a pathway for the production of the triplet state of the chromophore via internal conversion facilitated by oxygen. Our results provide concrete support to previously hypothesized scenarios.

The photochemically active sites of the proteins sfGFP66azF and Venus66azF, members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue p-azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by the formation of phenylamine-containing chromophores. We report the first study of the reaction mechanism of the reduction of the arylnitrene intermediates in sfGFP66nF and Venus66nF using molecular modeling methods. The Gibbs energy profiles for the elementary steps of the chemical reaction in sfGFP66nF are computed using molecular dynamics simulations with quantum mechanics/molecular mechanics (QM/MM) potentials. Structures and energies along the reaction pathway in Venus66nF are evaluated using a QM/MM approach. According to the results of the simulations, arylnitrene reduction is coupled with oxidation of the histidine side chain on the His148 residue located near the chromophore.

Gcn5-related N-acetyltransferases catalyze the transfer of an acetyl group to a primary amino group of a wide class of substrates. Deprotonation of the amino group upon binding to the enzyme is necessary to activate the nucleophilic attack on the substrate. The process of binding of glutamate to N-acetylglutamate synthase is considered using the methods of molecular modeling and quantum chemistry. It is shown that deprotonation of the primary amino group of glutamate occurs upon its incorporation into the active site of the enzyme with the participation of the side chain of the aspartate residue.

The search for efficient inhibitors of the SARS-CoV-2 enzymes is ongoing due to the continuing COVID-19 pandemic. We report the results of computational modeling of the reactions of the SARS-CoV-2 main protease (MPro ) with four potential covalent inhibitors. Two of them, carmofur and nirmatrelvir, have been shown experimentally the ability to inhibit MPro . Two other compounds, X77A and X77C, were designed computationally in this work, derived from the structure of X77, a non-covalent inhibitor forming a tight surface complex with MPro . We modified the X77 structure by introducing warheads capable of efficient chemical reactions with the catalytic cysteine residue in the M Pro active site. The reaction mechanisms of the four molecules with M Pro were investigated by quantum mechanics/molecular mechanics (QM/MM) calculations using large quantum subsystems. First, at the QM/MM level, we optimized structures of stationary points on the potential energy surfaces corresponding to the reactants, products, intermediates, and transition states along the hypothesized reaction coordinates. Analysis of these structures has informed the selection of collective variables for the subsequent calculations of the Gibbs energy profiles using molecular dynamics simulations with QM/MM potentials (QM/MM MD). In these simulations, the QM part was treated by DFT with the PBE0 functional. The results show that all four compounds form covalent adducts with the catalytic cysteine Cys 145 of MPro . From the chemical perspective, the reactions of these four compounds with M Pro follow three distinct mechanisms. In all cases, the reaction is initiated by a nucleophilic attack of the thiolate group of the deprotonated cysteine residue from the catalytic dyad Cys145-His41 of MPro . In the case of carmofur and X77A, the covalent binding of the thiolate to the ligand is accompanied by the formation of the fluoro-uracil leaving group. The reaction with X77C follows the nucleophilic aromatic substitution SN Ar mechanism. The reaction of M Pro with nirmatrelvir, which has a reactive nitrile group, leads to the formation of the covalent thioimidate adduct with the thiolate of the Cys145 residue in the enzyme active site.

Minima on the potential energy surface obtained at the QM(PBE0-D3/6-31G*)/MM(AMBER) level. Th reaction path is REAC ->INT1 -> INT2 -> PROD

Free energy profiles along the reaction coordinate were analyzed for several model systems by modeling elementary processes of the catalytic cycle of enzymes. A substantial contribution of fast non-limiting steps, which are conformational changes/adjustments, and proton transfer steps to the implementation of the catalytic cycle and high rates of enzymatic reactions is shown. The rate constants of conformational changes and proton transfer steps were determined from an analysis of the free energy profiles. In most cases, the lifetimes of the conformers lie in a range of 10 −10 s. A distinctive feature of enzymatic catalysis is the multistep character of the reactions, while the catalytic cycle is accomplished in the conformationally flexible polymer matrix with the conformational adjustment of the active site of the enzyme.

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.

We report the results of a computational study of the mechanism of the light-induced chemical reaction of chromophore hydration in the fluorescent protein Dreiklang, responsible for its switching from the fluorescent ON-state to the dark OFF-state. We explore the relief of the charge-transfer excited-state potential energy surface in the ON-state to locate minimum energy conical intersection points with the ground-state energy surface. Simulations of the further evolution of model systems allow us to characterize the ground-state reaction intermediate tentatively suggested in the femtosecond studies of the light-induced dynamics in Dreiklang and finally to arrive at the reaction product. The obtained results clarify the details of the photoswitching mechanism in Dreiklang, which is governed by the chemical modification of its chromophore.

Oxygenase activity of the flavin-dependent enzyme RutA is commonly associated with the formation of flavin-oxygen adducts in the enzyme active site. We report the results of quantum mechanics/molecular mechanics (QM/MM) modeling of possible reaction pathways initiated by various triplet state complexes of the molecular oxygen with the reduced flavin mononucleotide (FMN) formed in the protein cavities. According to the calculation results, these triplet-state flavin-oxygen complexes can be located at both re-side and si-side of the isoalloxazine ring of flavin. In both cases, the dioxygen moiety is activated by electron transfer from FMN, stimulating the attack of the arising reactive oxygen species at the C4a, N5, C6, and C8 positions in the isoalloxazine ring after the switch to the singlet state potential energy surface. The reaction pathways lead to the C(4a)-peroxide, N(5)-oxide, or C(6)-hydroperoxide covalent adducts or directly to the oxidized flavin, depending on the initial position of the oxygen molecule in the protein cavities.

The mechanism of the reactivation reaction of the double mutant butyrylcholinesterase (BChE) double mutantAsn322Glu/Glu325Gly, inhibited by the organophosphorus compound (OPC) echothiophat is studied by molecular modeling methods. The ability of this mutant to spontaneously reactivate itself was previously shown experimentally. The energy profile of the reaction path calculated by the method of quantum mechanics/molecular mechanics (QM/MM) confirms that such a mechanism is possible. Molecular dynamics calculations with QM/MM potentials are used to investigate the proton transfer pathways and determine the protonated state of the glutamic acids in the active site of the double mutant. Possible ways of increasing its conformational stability are investigated by the methods of classical molecular dynamics.

This work explores the level of transparency in reporting the details of computational protocols that is required for practical reproducibility of quantum mechanics/molecular mechanics (QM/MM) simulations. Using the reaction of an essential SARS-CoV-2 enzyme (the main protease) with a covalent inhibitor (carmofur) as a test case of chemical reactions in biomolecules, we carried out QM/MM calculations to determine the structures and energies of the reactants, the product, and the transition state/intermediate using analogous QM/MM models implemented in two software packages, NWChem and Q-Chem. Our main benchmarking goal was to reproduce the key energetics computed with the two packages. Our results indicate that quantitative agreement (within the numerical thresholds used in calculations) is difficult to achieve. We show that rather minor details of QM/ MM simulations must be reported in order to ensure the reproducibility of the results and offer suggestions toward developing practical guidelines for reporting the results of biosimulations.

Supercomputer molecular modeling methods are applied to characterize structure and dynamics of the flavin-dependent enzyme RutA in the complex with molecular oxygen. Following construction of a model protein system, molecular dynamics (MD) simulations were carried out using either classical force field interaction potentials or the quantum mechanics/molecular mechanics (QM/MM) potentials. Several oxygen-binding pockets in the protein cavities were located in these simulations. The QM/MM-based MD calculations rely on the interface between the quantum chemistry package TeraChem and the MD package NAMD. The results show a stable localization of the oxygen molecule in the enzyme active site. Static QM/MM calculations carried out with two different packages, NWChem and TURBOMOLE, allowed us to establish the structure of the RutA-O2 complex. Biochemical perspectives of the hallmark reaction of incorporating oxygen into organic compounds emerged from these simulations are formulated.

The results of a computational study of the synthesis of a key brain metabolite, N-acetyl-l-aspartate, catalyzed by aspartate N-acetyltransferase, encoded by the NAT8L gene, are reported. The reaction Gibbs energy profiles were computed using molecular dynamics simulations with interaction potentials estimated on-the-fly by the quantum mechanics/molecular mechanics QM(PBE0/6-31G**)/MM(CHARMM) approach. The revealed reaction mechanism includes four elementary steps with corresponding activation energies not exceeding 14 kcal mol−1