On CO Egress from,and Re‐Uptake by,the Enzyme MauG,as a Mimic of the Acquisition of Oxidizing Agents by the pre‐MADH_MauG System. A Molecular Mechanics Approach

In this work, by applying a non‐deterministic, randomly‐oriented minimal force to the dissociated CO ligand of the MauG‐CO system, the molecular‐dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high‐spin c‐heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H₂O molecules, CO was observed to reverse direction, re‐entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations.     

On CO Egress from, and Re-Uptake by, the Enzyme MauG, as a Mimic of the Acquisition of Oxidizing Agents by the pre-MADH_MauG System. A Molecular Mechanics Approach

In this work, by applying a non-deterministic, randomly-oriented minimal force to the dissociated CO ligand of the MauG-CO system, the molecular-dynamics (MD) behavior of this system could be quickly unraveled. It turned out that CO has no marked directional egress from the high-spin c-heme iron distal pocket. Rather, CO is able to exploit all interstices created during the protein fluctuations. Nonetheless, no steady route toward the surrounding solvent was ever observed: CO jumped first into other binding pockets before being able to escape the protein. In a few cases, on hitting the surrounding H(2) O molecules, CO was observed to reverse direction, re-entering the protein. A contention that conformational inversion of the P107 ring provides a gate to the iron ion is not supported by the present simulations.     

On the Pathways for CO Egress from Carboxy Human Cytoglobin. A Molecular‐Dynamics Investigation

This work discloses two bona fide gates through which the CO ligand can leave the distal cavity of carboxy human cytoglobin, reaching the solvent. The investigation was based on molecular dynamics, aided by a minimal randomly‐oriented force applied to the ligand. The shortest pathway progresses toward the main gate, H81‐R84, in the open state, with the H81 imidazole moiety turned toward the solvent. A longer pathway develops toward the diametrically opposed W31‐W151 gate. In between, CO may be entrapped into binding cavities, either along the path toward the gates, or in a cul‐de‐sac, from which CO may even be incapable to escape. This behavior contrasts with carboxy myoglobin, where the corresponding H64 gate, when opened, is the sole used by CO to get to the solvent. These observations, which could hold also for other small ligands of biological interest, such as O₂, NO, and NO$\rm{{_{3}^{-}}}$ , provide an answer to a neglected aspect of the mysterious six‐coordinated globins.     

Molecular‐Dynamics Simulation of Dioxygen Egress from 12/15‐Lipoxygenase?Arachidonic Acid Complex

Extensive random‐acceleration molecular‐dynamics (RAMD) simulations of the egress of dioxygen (O₂) from a model of rabbit 12/15‐lipoxygenase? arachidonic acid complex disclosed several exit portals in addition to those previously described from implicit ligand sampling calculations and limited MD simulations.     

Understanding How H‐NOX (Heme Nitric Oxide/Oxygen) Domain Works Needs First Clarifying How Diatomic Gases Are Relocated Inside This Sensing Protein. A Molecular‐Mechanics Approach

H‐NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H‐NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme‐dissociated CO of Nostoc sp. H‐NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H‐NOX with Xe gas. What is the main pathway with Xe‐cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature.     

Molecular-dynamics simulation of dioxygen egress from 12/15-lipoxygenase-arachidonic acid complex

Extensive random-acceleration molecular-dynamics (RAMD) simulations of the egress of dioxygen (O?) from a model of rabbit 12/15-lipoxygenase-arachidonic acid complex disclosed several exit portals in addition to those previously described from implicit ligand sampling calculations and limited MD simulations.     

Unveiling the Pathways of Dioxygen Through the C2 Component of the Environmentally Relevant Monooxygenase p‐Hydroxyphenylacetate Hydroxylase from Acinetobacter baumannii: A Molecular Dynamics Investigation

In this work, models of the homotetrameric C2 component of the monooxygenase p‐hydroxyphenylacetate hydroxylase from Acinetobacter baumannii, in complex with dioxygen (O₂) and, or not, the substrate p‐hydroxyphenylacetate (HPA) were built. Both models proved to be amenable to random‐acceleration molecular dynamics (RAMD) simulations, whereby a tiny randomly oriented external force, acting on O₂ at the active site in front of flavin mononucleotide (FMNH^?), accelerated displacement of O₂ toward the bulk solvent. This allowed us to carry out a sufficiently large number of RAMD simulations to be of statistical significance. The two systems behaved very similarly under RAMD, except for O₂ leaving the active site more easily in the absence of HPA, but then finding similar obstacles in getting to the gate as when the active site was sheltered by HPA. This challenges previous conclusions that HPA can only reach the active center after that the C4aOOH derivative of FMNH^? is formed, requiring uptake of O₂ at the active site before HPA. According to these RAMD simulations, O₂ could well get to FMNH^? also in the presence of the substrate at the active site.     

Retinal release from opsin in molecular dynamics simulations

The crystal structures of opsin in the ligand-free and the G-protein-interacting states showed two inter-helical openings between transmembrane (TM) helices TM1 and TM7 and between TM5 and TM6 near the extracellular side that were thought to serve as the retinal uptake and release gates. However, it is unclear which opening is for 11-cis-retinal uptake or all-trans-retinal release although speculations have been proposed based on the structural features of opsin and retinal. In this work, we simulated the exit process of all-trans-retinal from the ligand-free opsin structure by the classical molecular dynamics (MD) and random acceleration molecular dynamics (RAMD). In the 64 ns classical MD simulation, retinal remained in the receptor but moved significantly toward the TM5-TM6 opening and almost inserted into the opening after 50 ns. Complete exit was observed in 114 out of 160 RAMD trajectories with the TM5-TM6 opening being the predominant egress gate while egress from the TM1-TM7 opening was observed in only a few trajectories when relatively large acceleration was applied and large structural alteration of the protein resulted. These results suggest that photolyzed all-trans-retinal is likely released through the TM5-TM6 opening. Based on the unidirectional mechanism of retinal exchange suggested by experiment, we speculate that the TM1-TM7 opening serves as the 11-cis-retinal uptake gate. The spatial occupancy maps of retinal computed from the 160 RAMD trajectories further indicated that retinal experienced significant interactions with the receptor during the exit process. The implications of these findings for disease mechanisms of rhodopsin mutants are discussed.     

Binding Pockets and Permeation Channels for Dioxygen through Cofactorless 3‐Hydroxy‐2‐methylquinolin‐4‐one 2,4‐Dioxygenase in Association with Its Natural Substrate, 3‐Hydroxy‐2‐methylquinolin‐4(1H)‐one. A Perspective from Molecular Dynamics Simulations

This work describes an investigation of pathways and binging pockets (BPs) for dioxygen (O₂) through the cofactorless oxygenase 3‐hydroxy‐2‐methylquinolin‐4‐one 2,4‐dioxygenase in complex with its natural substrate, 3‐hydroxy‐2‐methylquinolin‐4(1H)‐one, in aqueous solution. The investigation tool was random‐acceleration molecular dynamics (RAMD), whereby a tiny, randomly oriented external force is applied to O₂ in order to accelerate its movements. In doing that, care was taken that the external force only continues, if O₂ moves along a direction for a given period of time, otherwise the force changed direction randomly. Gates for expulsion of O₂ from the protein, which can also be taken as gates for O₂ uptake, were found throughout almost the whole external surface of the protein, alongside a variety of BPs for O₂. The most exploited gates and BPs were not found to correspond to the single gate and BP proposed previously from the examination of the static model from X‐ray diffraction analysis of this system. Therefore, experimental investigations of this system that go beyond the static model are urgently needed.     

Gates and Binding Pockets for Nitric Oxide with Cytochrome c′, According to Molecular Dynamics

Random‐acceleration molecular‐dynamics (RAMD) simulations with models of homodimeric 6‐ligated distal‐NO and 5‐ligated proximal‐NO cytochrome c′ complexes, in TIP3 H₂O, showed two distinct, non‐intercommunicating worlds. In the framework of a long cavity formed by four protein helices with heme at one extremity, NO was observed to follow different pathways with the two complexes to reach the solvent. With the 6‐ligated complex, NO was observed to progress by exploiting protein internal channels created by thermal fluctuations, and be temporarily trapped into binding pockets before reaching the preferred gate at the heme end of the cavity. In contrast, with the 5‐ligated complex, NO was observed to surface the solvent‐exposed helix 7, up to a gate at the other extremity of the protein, only occasionally finding an earlier, direct way out toward the solvent. That only bulk NO gets involved in forming the 5‐ligated proximal‐NO complex is in agreement with previous experimental observations, while the occurrence of binding pockets suggests that also reservoir NO might play a role with the distal‐NO complex.     

Understanding How H-NOX (Heme Nitric Oxide/Oxygen) domain works needs first clarifying how diatomic gases are relocated inside this sensing protein. A molecular-mechanics approach

H-NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H-NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme-dissociated CO of Nostoc sp. H-NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H-NOX with Xe gas. What is the main pathway with Xe-cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature.     

On the Quest of Dioxygen by Monomeric Sarcosine Oxidase. A Molecular Dynamics Investigation

It is reported here on random acceleration molecular dynamics (RAMD) simulations with the 2GF3 bacterial monomeric sarcosine oxidase (MSOX), O₂, and furoic acid in place of sarcosine, solvated by TIP3 H₂O in a periodic box. An external tiny force, acting randomly on O₂, accelerated its relocation, from the center of activation between residue K265 and the si face of the flavin ring of the flavin adenine dinucleotide cofactor, to the surrounding solvent. Only three of the four O₂ gates previously described for this system along a composite method technique were identified, while two more major O₂ gates were found. The RAMD simulations also revealed that the same gate can be reached by O₂ along different pathways, often involving traps for O₂. Both the residence time of O₂ in the traps, and the total trajectory time for O₂ getting to the solvent, could be evaluated. The new quick pathways discovered here suggest that O₂ exploits all nearby interstices created by the thermal fluctuations of the protein, not having necessarily to look for the permanent large channel used for uptake of the FADH cofactor. To this regard, MSOX resembles closely KijD3 N‐oxygenase. These observations solicit experimental substantiation, in a long term aim at discovering whether gates and pathways for the small gaseous ligands inside the proteins are under Darwinian functional evolution or merely stochastic control operates.     

Structural dynamics of distal histidine replaced mutants of myoglobin accompanied with the photodissociation reaction of the ligand

Protein dynamics observed by the transient grating (TG) method are studied for some site-directed mutants at the distal histidine of myoglobin (H64L, H64Q, H64V). The time profiles of the TG signals are very sensitive to the amino acid residue of the 64 position. It was found that the sensitivity is mostly caused by the different rates of the ligand escape from the protein to solvent and the magnitude of the molecular volume change. Several molecular origins of the volume difference between MbCO and Mb, such as the electrostatic interaction in the distal pocket, movement of helices, and distal water, are proposed. Interestingly, the volume difference between the CO-trapped Mb inside the protein interior and Mb is similar to that of the partial molar volume of CO in organic solvent. The effect of mutation on the nature of the CO trapped site is discussed.     

On Dioxygen Permeation of MaL Laccase from the Thermophilic Fungus Melanocarpus albomyces: An all‐Atom Molecular Dynamics Investigation

In this article, biased molecular dynamics (MD) simulations of O₂ egress from the active center of MaL laccase toward the bulk solvent were described. Parameterization of the set of four Cu(II) ions, in the framework of CHARMM‐36 FF, was carried out on a recent dummy‐atom model that takes into account the Jahn–Teller effect. By carrying out a number of statistically relevant MD simulations, under a tiny randomly oriented external force applied to the molecule O₂, three preferred gates for O₂ egress from the enzyme and a few intermediate binding pockets (BPs) for O₂ were visible; all the gates and pockets were located on two of the three domains. This wide distribution of preferred gates notwithstanding the molecule O₂ was seen to follow specific pathways, exploiting consistently the interstices created by the enzyme thermal fluctuations. These are features that can be imagined to have evolved to make MaL laccase extremely efficient as catalysts in various reactions that require O₂.     

New Vistas on the Recruiting of Ferrous Iron and Dioxygen by Ferritins: A Case Study of the Escherichia coli 24‐mer Ferritin by All‐Atom Molecular Dynamics in Aqueous Medium

It is shown here that Fe²⁺ and O₂ ligands are displaced from the ferroxidase center of the C1 four‐helix bundle of E. coli 24‐mer ferritin under molecular dynamics (MD) aided by a randomly oriented external force applied to the ligand. Under these conditions, ligand egress toward the external aqueous medium occurs preferentially from the same four‐helix bundle, in the case of O₂, or other bundle, in the case of Fe². Viewing ligand egress from the protein as the microscopic reverse of ligand influx into the protein under unbiased MD, these findings challenge current views that preferential gates for recruitment of Fe²⁺ are 3‐fold channels with human ferritin, or the short path from the ferroxidase center to H93 with bacterial ferritins.     

A molecular dynamics simulation of the flavin mononucleotide–RNA aptamer complex

We report on an unrestrained molecular dynamics simulation of the flavin mononucleotide (FMN)–RNA aptamer. The simulated average structure maintains both cross‐strand and intermolecular FMN–RNA nuclear Overhauser effects from the nmr experiments and has all qualitative features of the nmr structure including the G10–U12–A25 base triple and the A13–G24, A8–G28, and G9–G27 mismatches. However, the relative orientation of the hairpin loop to the remaining part of the molecule differs from the nmr structure. The simulation predicts that the flexible phosphoglycerol part of FMN moves toward G27 and forms hydrogen bonds. There are structurally long‐lived water molecules in the FMN binding pocket forming hydrogen bonds within FMN and between FMN and RNA. In addition, long‐lived water is found bridging primarily RNA backbone atoms. A general feature of the environment of long‐lived “structural” water is at least two and in most cases three or four potential acceptor atoms. The 2′‐OH group of RNA usually acts as an acceptor in interactions with the solvent. There are almost no intrastrand O2′H(n)⋮O4′(n + 1) hydrogen bonds within the RNA backbone. In the standard case the preferred orientation of the 2′‐OH hydrogen atoms is approximately toward O3′ of the same nucleotide. However, a relatively large number of conformations with the backbone torsional angle γ in the trans orientation is found. A survey of all experimental RNA x‐ray structures shows that this backbone conformation occurs but is less frequent than found in the simulation. Experimental nmr RNA aptamer structures have a higher fraction of this conformation as compared to the x‐ray structures. The backbone conformation of nucleotide n + 1 with the torsional angle γ in the trans orientation leads to a relatively short distance between 2′‐OH(n) and O5′(n + 1), enabling hydrogen‐bond formation. In this case the preferred orientation of the 2′‐OH hydrogen atom is approximately toward O5′(n + 1). We find two relatively short and dynamically stable types of backbone–backbone next‐neighbor contacts, namely C2′(H)(n)⋮O4′(n + 1) and C5′(H)(n + 1)⋮O2′(n). These interactions may affect both backbone rigidity and thermodynamic stability of RNA helical structures. © 1999 John Wiley & Sons, Inc. Biopoly 50: 287–302, 1999     

Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.     

The distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of two distal histidine tautomers.

Four independent 90 ps molecular dynamics simulations of sperm-whale wild-type carbonmonoxy myoglobin (MbCO) have been calculated using a new AMBER force field for the haem prosthetic group. Two trajectories have the distal 64N delta nitrogen protonated, and two have the 64N epsilon nitrogen protonated; all water molecules within 16 A of the carbonyl O are included. In three trajectories, the distal residue remains part of the haem pocket, with the protonated distal nitrogen pointing into the active site. This is in contrast with the neutron diffraction crystal structure, but is consistent with the solution phase CO stretching frequencies (upsilon CO) of MbCO and various of its mutants. There are significant differences in the "closed" pocket structures found for each tautomer: the 64N epsilon H trajectories both show stable distal-CO interactions, whereas the 64N delta H tautomer) has a weaker interaction resulting in a more mobile distal side chain. One trajectory (a 64N delta H tautomer) has the distal histidine moving out into the "solvent", leaving the pocket in an "open" structure, with a large unhindered entrance to the active site. These trajectories suggest that the three upsilon CO frequencies observed for wild-type MbCO in solution, rather than representing significantly different Fe-C-O geometries as such, arise from three different haem pocket structures, each with different electric fields at the ligand. Each pocket structure corresponds to a different distal histidine conformer: the A3 band to the 64N epsilon H tautomer, the A1,2 band to the 64N delta H tautomer, and the A0 band to the absence of any significant interaction with the distal side chain.     

Restoring Histone Deacetylase Activity by Waste Product Release. A View from Molecular Mechanics Simulations with Mammalian HDAC8

HDAC8 is a Zn^II‐based, single‐peptide mammalian histone deacetylase that is localized mainly in the cytoskeleton of smooth muscle cells, thus regulating muscle contractility. HDACs are also widely involved in cellular processes, ranging from cell differentiation to proliferation, senescence, and apoptosis; in particular, protecting a telomerase activator from ubiquitin‐mediated degradation. How HDACs can eliminate the hydrolytic reaction products, in order that the process of deacetylation of the acetyllysine moiety of histones can take place again, has long been debated in the scientific literature, without reaching any firm conclusion, however. This question is the subject of the present work, carried out along a theoretical line that is capable of describing the whole pathway followed by the acetate product (ACT). A model was built here on the crystal data for the Y306F‐mutated HDAC8 complex with a diacetylated peptide of the p53‐tumor‐suppressor class. That was followed by manually hydrolyzing the acetylated moiety bound to Zn^II and discharging the monoacetylated peptide product (MAP). The latter was replaced by a H₂O molecule bound to Zn^II, while ACT was left free in the reaction cage. This Zn^II cluster was DFT‐parameterized for the ff99SB force field without any further bias. As the result of random‐acceleration molecular dynamics (RAMD) simulations, egress of ACT from the reaction cage toward the aqueous environment can follow three pathways. Two of them utilize the channel for peptide (or histone) uptake and are preferred, if ACT leaves the reaction center before MAP (or the deacetylated histone). The third pathway, developing along the internal channel, is available to ACT even if MAP is still in place.     

On the Permeation by Dioxygen of the Cofactor‐Independent Unusual Oxygenase RhCC,in Complex with Substrate 4‐Hydroxyphenylenolpyruvate. A Molecular Dynamics Investigation

This work deals with a trimeric bacterial protein, RhCC, which, although belonging to the tautomerase superfamily, shows oxygenase activity. A model of the complex from RhCC and substrate 4‐hydroxyphenylenolpyruvate (4HPP), fitting the observation of extra electron densities from X‐ray diffraction of the crystal, could be built by autodocking. When subjected to molecular dynamics (MD) aided by an external random force applied to a O₂ molecule placed above 4HPP, this model evolved with O₂ egressing toward the bulk solvent from two nearly opposite gates. These were located between the nearly parallel helices 75 – 91 and 15 – 33 of either chain C (gate SE) or chain B (gate FL). Alternatively, with four O₂ molecules in the bulk solvent, unbiased MD led to O₂ entering the protein from gate SE and getting to 4HPP, while forming a stabilizing salt bridge between the 4HPP carboxylate and P1.C ⁺NH₂, thus providing scientific ground for a refined model of the complex.