Molecular dynamics simulation of the last step of a catalytic cycle: Product release from the active site of the enzyme chorismate mutase from Mycobacterium tuberculosis

The protein chorismate mutase MtCM from Mycobacterium tuberculosis catalyzes one of the few pericyclic reactions known in biology: the transformation of chorismate to prephenate. Chorismate mutases have been widely studied experimentally and computationally to elucidate the transition state of the enzyme catalyzed reaction and the origin of the high catalytic rate. However, studies about substrate entry and product exit to and from the highly occluded active site of the enzyme have to our knowledge not been performed on this enzyme. Crystallographic data suggest a possible substrate entry gate, that involves a slight opening of the enzyme for the substrate to access the active site. Using multiple molecular dynamics simulations, we investigate the natural dynamic process of the product exiting from the binding pocket of MtCM. We identify a dominant exit pathway, which is in agreement with the gate proposed from the available crystallographic data. Helices H2 and H4 move apart from each other which enables the product to exit from the active site. Interestingly, in almost all exit trajectories, two residues arginine 72 and arginine 134, which participate in the burying of the active site, are accompanying the product on its exit journey from the catalytic site.     

Ligand-release pathways in the pheromone-binding protein of Bombyx mori

Pheromone-binding proteins (PBP) supply olfactory neuron cells with pheromones by binding the ligands they are tailored for and carrying them to their receptor. The function of a PBP as an efficient carrier requires fast ligand uptake and release. The molecular basis of the ligand-binding mechanism was addressed here for the intriguing case of the PBP of the silk moth Bombyx mori. This PBP completely encapsulates its ligand bombykol without displaying any obvious ligand entrance/exit sites. Here, two opposite dissociation routes were identified as the most likely entrance/exit paths by replica-exchange molecular dynamics, essential dynamics, and force-probe molecular dynamics simulations. One of the paths runs along a flexible front lid; the other along the termini at the back. Calculated forces and energies suggest that both routes are physiologically relevant. The multiplicity of pathways may reduce or tune the entropic barrier for ligand binding.     

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg²⁺) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.     

How an Inhibitor Bound to Subunit Interface Alters Triosephosphate Isomerase Dynamics

The tunnel region at triosephosphate isomerase (TIM)’s dimer interface, distant from its catalytic site, is a target site for certain benzothiazole derivatives that inhibit TIM’s catalytic activity in Trypanosoma cruzi, the parasite that causes Chagas disease. We performed multiple 100-ns molecular-dynamics (MD) simulations and elastic network modeling (ENM) on both apo and complex structures to shed light on the still unclear inhibitory mechanism of one such inhibitor, named bt10. Within the time frame of our MD simulations, we observed stabilization of aromatic clusters at the dimer interface and enhancement of intersubunit hydrogen bonds in the presence of bt10, which point to an allosteric effect rather than destabilization of the dimeric structure. The collective dynamics dictated by the topology of TIM is known to facilitate the closure of its catalytic loop over the active site that is critical for substrate entrance and product release. We incorporated the ligand’s effect on vibrational dynamics by applying mixed coarse-grained ENM to each one of 54,000 MD snapshots. Using this computationally efficient technique, we observed altered collective modes and positive shifts in eigenvalues due to the constraining effect of bt10 binding. Accordingly, we observed allosteric changes in the catalytic loop’s dynamics, flexibility, and correlations, as well as the solvent exposure of catalytic residues. A newly (to our knowledge) introduced technique that performs residue-based ENM scanning of TIM revealed the tunnel region as a key binding site that can alter global dynamics of the enzyme.     

How do substrates enter and products exit the buried active site of cytochrome P450cam? 1. Random expulsion molecular dynamics investigation of ligand access channels and mechanisms

Cytochrome P450s form a ubiquitous protein family with functions including the synthesis and degradation of many physiologically important compounds and the degradation of xenobiotics. Cytochrome P450cam from Pseudomonas putida has provided a paradigm for the structural understanding of cytochrome P450s. However, the mechanism by which camphor, the natural substrate of cytochrome P450cam, accesses the buried active site is a long-standing puzzle. While there is recent crystallographic and simulation evidence for opening of a substrate-access channel in cytochrome P450BM-3, for cytochrome P450cam, no such conformational changes have been observed either in different crystal structures or by standard molecular dynamics simulations. Here, a novel simulation method, random expulsion molecular dynamics, is presented, in which substrate-exit channels from the buried active site are found by imposing an artificial randomly oriented force on the substrate, in addition to the standard molecular dynamics force field. The random expulsion molecular dynamics method was tested in simulations of the substrate-bound structure of cytochrome P450BM-3, and then applied to complexes of cytochrome P450cam with different substrates and with product. Three pathways were identified, one of which corresponds to a channel proposed earlier on the basis of crystallographic and site-directed mutagenesis data. Exit via the water-filled channel, which was previously suggested to be a product exit channel, was not observed. The pathways obtained by the random expulsion molecular dynamics method match well with thermal motion pathways obtained by an analysis of crystallographic B-factors. In contrast to large backbone motions (up to 4 A) observed in cytochrome P450BM-3 for the exit of palmitoleic acid, passage of camphor through cytochrome P450cam only requires small backbone motions (less than 2.4 A) in conjunction with side-chain rotations. Concomitantly, in almost all the exit trajectories, salt-links that have been proposed to act as ionic tethers between secondary structure elements of the protein, are perturbed.     

Molecular dynamics of substrate complexes with hamster cytochrome P450c17 (CYP17): mechanistic approach to understanding substrate binding and activities

The cytochrome P450c17 isoforms from various animal species have different substrate selectivity, especially for 17,20-lyase activity. In particular, the human P450c17 selectively produces dehydroepiandrosterone with little androstenedione (AD). Hamster P450c17, on the other hand, produces both of these steroids at comparable rates. We thus investigated if computational analysis could explain the difference in activity profiles. Therefore, we inserted the four P450c17 substrates-pregnenolone, progesterone, and their 17alpha-hydroxylated forms-inside our hamster P450c17 model, which we derived from our human P450c17 model based on the crystal structure of P450BMP. We performed molecular dynamics (MD) simulations on the complexes and analyzed the resultant trajectories to identify amino acids that interact with substrates. Starting with substrates in two different orientations, we obtained two sets of binding trajectories in each case. The first set of trajectories reveal structural rearrangements that occur during binding, whereas the second set of trajectories reflects substrate orientations during catalysis. Our modeling suggests that three distinct steps are required for substrate selectivity and binding to the hamster P450c17: (1) recognition of the substrate at the putative substrate entrance, characterized by a pocket at the surface of the hamster P450c17 containing charged residues R96 and D116; (2) entry of the substrate into the active site, in an intermediate position directed by possible hydrogen bonding of the substrates with the heme D-ring propionate group, R96, R440, and T306; followed by (3) 90 degrees counterclockwise rotation of the substrates, positioning them in optimal position for reactivity, a process that may be directed by hydrogen bonding to the 110-112 region of the hamster P450c17. With some substrates, we obtained trajectories which suggest that major distortions in the I-helix and opening of the H-I loop occur during substrate binding. In conclusion, these modeling exercises provide insight to possible structural reorganizations that occur during substrate binding and suggest that amino acids that participate in three distinct steps of this process may all contribute to substrate binding and activity.     

Theoretical Investigations of Nitric Oxide Channeling in Mycobacterium tuberculosis Truncated Hemoglobin N

Mycobacterium tuberculosis group I truncated hemoglobin trHbN catalyzes the oxidation of nitric oxide (•NO) to nitrate with a second-order rate constant k ≈ 745 μM⁻¹ s⁻¹ at 23°C (nitric oxide dioxygenase reaction). It was proposed that this high efficiency is associated with the presence of hydrophobic tunnels inside trHbN structure that allow substrate diffusion to the distal heme pocket. In this work, we investigated the mechanisms of •NO diffusion within trHbN tunnels in the context of the nitric oxide dioxygenase reaction using two independent approaches. Molecular dynamics simulations of trHbN were performed in the presence of explicit •NO molecules. Successful •NO diffusion from the bulk solvent to the distal heme pocket was observed in all simulations performed. The simulations revealed that •NO interacts with trHbN at specific surface sites, composed of hydrophobic residues located at tunnel entrances. The entry and the internal diffusion of •NO inside trHbN were performed using the Long, Short, and EH tunnels reported earlier. The Short tunnel was preferentially used by •NO to reach the distal heme pocket. This preference is ascribed to its hydrophobic funnel-shape entrance, covering a large area extending far from the tunnel entrance. This funnel-shape entrance triggers the frequent formation of solvent-excluded cavities capable of hosting up to three •NO molecules, thereby accelerating •NO capture and entry. The importance of hydrophobicity of entrances for •NO capture is highlighted by a comparison with a polar mutant for which residues at entrances were mutated with polar residues. A complete map of •NO diffusion pathways inside trHbN matrix was calculated, and •NO molecules were found to diffuse from Xe cavity to Xe cavity. This scheme was in perfect agreement with the three-dimensional free-energy distribution calculated using implicit ligand sampling. The trajectories showed that •NO significantly alters the dynamics of the key amino acids of Phe⁶²(E15), a residue proposed to act as a gate controlling ligand traffic inside the Long tunnel, and also of Ile¹¹⁹(H11), at the entrance of the Short tunnel. It is noteworthy that •NO diffusion inside trHbN tunnels is much faster than that reported previously for myoglobin. The results presented in this work shed light on the diffusion mechanism of apolar gaseous substrates inside protein matrix.     

Ligand unbinding pathways from the vitamin D receptor studied by molecular dynamics simulations

Molecular dynamics simulation techniques have been used to study the unbinding pathways of 1α,25-dihydroxyvitamin D₃ from the ligand-binding pocket of the vitamin D receptor (VDR). The pathways observed in a large number of relatively short (<200 ps) random acceleration molecular dynamics (RAMD) trajectories were found to be in fair agreement, both in terms of pathway locations and deduced relative preferences, compared to targeted molecular dynamics (TMD) and streered molecular dynamics simulations (SMD). However, the high-velocity ligand expulsions of RAMD tend to favor straight expulsion trajectories and the observed relative frequencies of different pathways were biased towards the probability of entering a particular exit channel. Simulations indicated that for VDR the unbinding pathway between the H1–H2 loop and the β-sheet between H5 and H6 is more favorable than the pathway located between the H1–H2 loop and H3. The latter pathway has been suggested to be the most likely unbinding path for thyroid hormone receptors (TRs) and a likely path for retinoic acid receptor. Ligand entry/exit through these two pathways would not require displacement of H12 from its agonistic position. Differences in the packing of the H1, H2, H3 and β-sheet region explain the changed relative preference of the two unbinding pathways in VDR and TRs. Based on the crystal structures of the ligand binding domains of class 2 nuclear receptors, whose members are VDR and TRs, this receptor class can be divided in two groups according to the packing of the H1, H2, H3 and β-sheet region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A Wrench in the Works of Human Acetylcholinesterase: Soman Induced Conformational Changes Revealed by Molecular Dynamics Simulations

Irreversible inactivation of human acetylcholinesterase (hAChE) by organophosphorous pesticides (OPs) and chemical weapon agents (CWA) has severe morbidity and mortality consequences. We present data from quantum mechanics/molecular mechanics (QM/MM) and 80 classical molecular dynamics (MD) simulations of the apo and soman-adducted forms of hAChE to investigate the effects on the dynamics and protein structure when the catalytic Serine 203 is phosphonylated. We find that the soman phosphonylation of the active site Ser203 follows a water assisted addition-elimination mechanism with the elimination of the fluoride ion being the highest energy barrier at 6.5 kcal/mole. We observe soman-dependent changes in backbone and sidechain motions compared to the apo form of the protein. These alterations restrict the soman-adducted hAChE to a structural state that is primed for the soman adduct to be cleaved and removed from the active site. The altered motions and resulting structures provide alternative pathways into and out of the hAChE active site. In the soman-adducted protein both side and back door pathways are viable for soman adduct access. Correlation analysis of the apo and soman adducted MD trajectories shows that the correlation of gorge entrance and back door motion is disrupted when hAChE is adducted. This supports the hypothesis that substrate and product can use two different pathways as entry and exit sites in the apo form of the protein. These alternative pathways have important implications for the rational design of medical countermeasures.     

The structure of monoacylglycerol lipase from Bacillus sp. H257 reveals unexpected conservation of the cap architecture between bacterial and human enzymes

Monoacylglycerol lipases (MGLs) catalyse the hydrolysis of monoacylglycerol into free fatty acid and glycerol. MGLs have been identified throughout all genera of life and have adopted different substrate specificities depending on their physiological role. In humans, MGL plays an integral part in lipid metabolism affecting energy homeostasis, signalling processes and cancer cell progression. In bacteria, MGLs degrade short-chain monoacylglycerols which are otherwise toxic to the organism. We report the crystal structures of MGL from the bacterium Bacillus sp. H257 (bMGL) in its free form at 1.2 Å and in complex with phenylmethylsulfonyl fluoride at 1.8 Å resolution. In both structures, bMGL adopts an α/β hydrolase fold with a cap in an open conformation. Access to the active site residues, which were unambiguously identified from the protein structure, is facilitated by two different channels. The larger channel constitutes the highly hydrophobic substrate binding pocket with enough room to accommodate monoacylglycerol. The other channel is rather small and resembles the proposed glycerol exit hole in human MGL. Molecular dynamics simulation of bMGL yielded open and closed states of the entrance channel and the glycerol exit hole. Despite differences in the number of residues, secondary structure elements, and low sequence identity in the cap region, this first structure of a bacterial MGL reveals striking structural conservation of the overall cap architecture in comparison with human MGL. Thus it provides insight into the structural conservation of the cap amongst MGLs throughout evolution and provides a framework for rationalising substrate specificities in each organism.     

Insights into substrate and product traffic in the Drosophila melanogaster acetylcholinesterase active site gorge by enlarging a back channel

To test a product exit differing from the substrate entrance in the active site of acetylcholinesterase (EC 3.1.1.7), we enlarged a channel located at the bottom of the active site gorge in the Drosophila enzyme. Mutation of Trp83 to Ala or Glu widens the channel from 5 A to 9 A. The kinetics of substrate hydrolysis and the effect of ligands that close the main entrance suggest that the mutations facilitate both product exit and substrate entrance. Thus, in the wild-type, the channel is so narrow that the 'back door' is used by at most 5% of the traffic, with the majority of traffic passing through the main entrance. In mutants Trp83Ala and Trp83Glu, ligands that close the main entrance do not inhibit substrate hydrolysis because the traffic can pass via an alternative route, presumably the enlarged back channel.     

Molecular modeling studies of substrate binding by penicillin acylase

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.     

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.     

Structural analysis of prolyl oligopeptidases using molecular docking and dynamics: insights into conformational changes and ligand binding

Prolyl oligopeptidase (POP) is considered as an important pharmaceutical target for the treatment of numerous diseases. Despite enormous studies on various aspects of POPs structure and function still some of the questions are intriguing like conformational dynamics of the protein and interplay between ligand entry/egress. Here, we have used molecular modeling and docking based approaches to unravel questions like differences in ligand binding affinities in three POP species (porcine, human and A. thaliana). Despite high sequence and structural similarity, they possess different affinities for the ligands. Interestingly, human POP was found to be more specific, selective and incapable of binding to a few planar ligands which showed extrapolation of porcine POP in human context is more complicated. Possible routes for substrate entry and product egress were also investigated by detailed analyses of molecular dynamics (MD) simulations for the three proteins. Trajectory analysis of bound and unbound forms of three species showed differences in conformational dynamics, especially variations in β-propeller pore size, which was found to be hidden by five lysine residues present on blades one and seven. During simulation, β-propeller pore size was increased by ∼2 Å in porcine ligand-bound form which might act as a passage for smaller product movement as free energy barrier was reduced, while there were no significant changes in human and A. thaliana POPs. We also suggest that these differences in pore size could lead to fundamental differences in mode of product egress among three species. This analysis also showed some functionally important residues which can be used further for in vitro mutagenesis and inhibitor design. This study can help us in better understanding of the etiology of POPs in several neurodegenerative diseases.     

Escape of a Small Molecule from Inside T4 Lysozyme by Multiple Pathways

The T4 lysozyme L99A mutant is often used as a model system to study small-molecule binding to proteins, but pathways for ligand entry and exit from the buried binding site and the associated protein conformational changes have not been fully resolved. Here, molecular dynamics simulations were employed to model benzene exit from its binding cavity using the weighted ensemble (WE) approach to enhance sampling of low-probability unbinding trajectories. Independent WE simulations revealed four pathways for benzene exit, which correspond to transient tunnels spontaneously formed in previous simulations of apo T4 lysozyme. Thus, benzene unbinding occurs through multiple pathways partially created by intrinsic protein structural fluctuations. Motions of several α-helices and side chains were involved in ligand escape from metastable microstates. WE simulations also provided preliminary estimates of rate constants for each exit pathway. These results complement previous works and provide a semiquantitative characterization of pathway heterogeneity for binding of small molecules to proteins.     

Binding of NAD+ and l-Threonine Induces Stepwise Structural and Flexibility Changes in Cupriavidus necator l-Threonine Dehydrogenase

Crystal structures of short chain dehydrogenase-like l-threonine dehydrogenase from Cupriavidus necator (CnThrDH) in the apo and holo forms were determined at 2.25 and 2.5 Å, respectively. Structural comparison between the apo and holo forms revealed that four regions of CnThrDH adopted flexible conformations when neither NAD⁺ nor l-Thr were bound: residues 38–59, residues 77–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations performed at the 50-ns time scale revealed that three of these regions remained flexible when NAD⁺ was bound to CnThrDH: residues 80–87, residues 180–186, and the catalytic domain. Molecular dynamics simulations also indicated that the structure of CnThrDH changed from a closed form to an open form upon NAD⁺ binding. The newly formed cleft in the open form may function as a conduit for substrate entry and product exit. These computational results led us to hypothesize that the CnThrDH reaction progresses by switching between the closed and open forms. Enzyme kinetics parameters of the L80G, G184A, and T186N variants also supported this prediction: the k_cat/K_{m, l-Thr} value of the variants was >330-fold lower than that of the wild type; this decrease suggested that the variants mostly adopt the open form when l-Thr is bound to the active site. These results are summarized in a schematic model of the stepwise changes in flexibility and structure that occur in CnThrDH upon binding of NAD⁺ and l-Thr. This demonstrates that the dynamical structural changes of short chain dehydrogenase-like l-threonine dehydrogenase are important for the reactivity and specificity of the enzyme.     

Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes.     

O2 Reactivity of Flavoproteins: DYNAMIC ACCESS OF DIOXYGEN TO THE ACTIVE SITE AND ROLE OF A H+ RELAY SYSTEM IN d-AMINO ACID OXIDASE*

Molecular dynamics simulations and implicit ligand sampling methods have identified trajectories and sites of high affinity for O₂ in the protein framework of the flavoprotein d-amino-acid oxidase (DAAO). A specific dynamic channel for the diffusion of O₂ leads from solvent to the flavin Si-side (amino acid substrate and product bind on the Re-side). Based on this, amino acids that flank the putative O₂ high affinity sites have been exchanged with bulky residues to introduce steric constraints. In G52V DAAO, the valine side chain occupies the site that in wild-type DAAO has the highest O₂ affinity. In this variant, the reactivity of the reduced enzyme with O₂ is decreased ≥100-fold and the turnover number ≈1000-fold thus verifying the concept. In addition, the simulations have identified a chain of three water molecules that might serve in relaying a H⁺ from the product imino acid =NH₂⁺ group bound on the flavin Re-side to the developing peroxide on the Si-side. This function would be comparable with that of a similarly located histidine in the flavoprotein glucose oxidase.     

Nitric Oxide Dynamics in Truncated Hemoglobin: Docking Sites, Migration Pathways, and Vibrational Spectroscopy from Molecular Dynamics Simulations

Atomistic simulations of nitric oxide (NO) dynamics and migration in the trHbN of Mycobacterium tuberculosis are reported. From extensive molecular dynamics simulations (48 ns in total), the structural and energetic properties of the ligand docking sites in the protein have been characterized and a connectivity network between the ligand docking sites has been built. Several novel migration and exit pathways are found and are analyzed in detail. The interplay between a hydrogen-bonding network involving residues Tyr³³ and Gln⁵⁸ and the bound O₂ ligand is discussed and the role of Phe⁶² residue in ligand migration is examined. It is found that Phe⁶² is directly involved in controlling ligand migration. This is reminiscent of His⁶⁴ in myoglobin, which also plays a central role in CO migration pathways. Finally, infrared spectra of the NO molecule in different ligand docking sites of the protein are calculated. The pocket-specific spectra are typically blue-shifted by 5-10 cm⁻¹, which should be detectable in future spectroscopic experiments.