Classical Molecular Dynamics

Cytochrome ba3 is a proton-pumping heme-copper oxygen reductase from the extreme thermophile Thermus thermophilus. Despite the fact that the enzyme's active site is buried deep within the protein, the apparent second order rate constant for the initial binding of O2 to the active-site heme has been experimentally found to be 10(9) M(-1) s(-1) at 298 K, at or near the diffusion limit, and 2 orders of magnitude faster than for O2 binding to myoglobin. To provide quantitative and microscopic descriptions of the O2 delivery pathway and mechanism in cytochrome ba3, extensive molecular dynamics simulations of the enzyme in its membrane-embedded form have been performed, including different protocols of explicit ligand sampling (flooding) simulations with O2, implicit ligand sampling analysis, and in silico mutagenesis. The results show that O2 diffuses to the active site exclusively via a Y-shaped hydrophobic tunnel with two 25-Å long membrane-accessible branches that coincide with th...

We highlight that machine-learning interatomic potentials trained over short AIMD trajectories enable first-principles multiscale modeling, bridging DFT level accuracy to the continuum level and empowering the study of complex/novel nanostructures.

We report a multi-scale simulation study of the photocycle of the rhodopsins. The quasi-atomistic representation ("united atoms" UA) of retinal is combined with a minimalist coarse grained (CG, one-bead-per amino acid) representation of the protein, in a hybrid UA/CG approach, which is the homolog of QM/MM, but at lower resolution. An accurate multi-stable parameterization of the model allows simulating each state and transition among them, and the combination of different scale representation allows addressing the entire photocycle. We test the model on bacterial rhodopsin, for which more experimental data are available, and then also report results for mammalian rhodopsins. In particular, the analysis of simulations reveals the spontaneous appearance of meta-stable states in quantitative agreement with experimental data.

We report a multi-scale simulation study of the photocycle of the rhodopsins. The quasi-atomistic representation ("united atoms" UA) of retinal is combined with a minimalist coarse grained (CG, one-bead-per amino acid) representation of the protein, in a hybrid UA/CG approach, which is the homolog of QM/MM, but at lower resolution. An accurate multi-stable parameterization of the model allows simulating each state and transition among them, and the combination of different scale representation allows addressing the entire photocycle. We test the model on bacterial rhodopsin, for which more experimental data are available, and then also report results for mammalian rhodopsins. In particular, the analysis of simulations reveals the spontaneous appearance of meta-stable states in quantitative agreement with experimental data.

The nanoindentation hardnesses and stacking fault energies (SFE) for pure and alloyed Au are determined from classical molecular dynamics simulations. Rather than a traditional force-displacement dependence that is examined in many previous nanoindentation works, we analyze the hardness vs. force in this study, which shows features that allow us to distinguish defect nucleation processes from hardening processes. During nanoindentation, homogeneously nucleated defects interact to form V-shape lock structures, and finally form four-sided dislocations that are continuously released into the bulk, in a manner similar to the

An interatomic model potential for molecular dynamics is derived from first-principles and used to study the molecular rotations and relaxation times in methylammonium lead halide, here considered the prototypical example of a hybrid crystal with a strong reorientational dynamics. Within the limits of a simple ionic scheme, the potential is able to catch the main qualitative features of the material at zero and finite temperature and opens the way to the development of classical potentials for hybrid perovskites. In agreement with experiments and previous theoretical findings the molecule trajectories exhibit a transition from a dynamics dominated by high symmetry directions at low temperature, to a fast dynamics at room temperature in which the molecule can reorient quasi-randomly. By computing the angular time correlation function we discuss the reorientational time as a function of temperature in comparison with existing literature providing a simple model and a clear attribution of the relaxation times in terms of their temperature dependence. This work clarifies the temperature dependence of the relaxation times and the interpretation of the experimental data in terms of the di↵erent mechanisms contributing to the molecules dynamics. Graphical TOC Entry Rotational relaxation times of methylammonium cations in hybrid lead halide perovskite are studied by classical molecular dynamics as a function of temperature

The kinetic properties of the ba 3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70°C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O 2 reaction, the decay of the F intermediate is significantly slower (k′) 100 s-1 at 5°C) than in the mitochondrial enzyme, with an activation energy E* of 10.1 (0.9 kcal mol-1. The cyanide-inhibited ba 3 oxidizes cyt c 522 quickly (k ≈ 5 × 10 6 M-1 s-1 at 25°C) and selectively, with an activation energy E* of 10.9 (0.9 kcal mol-1 , but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c 552 oxidase activity is enhanced up to 60°C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c 522-ba 3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba 3 for CO at 20°C (K eq) 1 × 10 5 M-1) was found to be smaller than that of beef heart aa 3 (K eq) 4 × 10 6 M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a 3 2+ of ba 3 (k′) 0.8 s-1 vs k′) 0.02 s-1 for beef heart aa 3 at 20°C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O 2 tensions is discussed.

The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O2, NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba3 from T. thermophilus, which is responsible for the activation and reduction of both O2 and NO, to gain insight into the structure of ligandbinding intermediates at ambient temperature. We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a3. The 2131 cm-1 mode of the "docked" CO we have detected corresponds to the B1 state of Mb and persists for 35 µs. The release of CO from the docking site is not followed by recombination to the heme a3 Fe. Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a3 propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a3 Fe for a few milliseconds. Rebinding to heme a3 occurs with k2) 29.5 s-1. These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.

Of the spectroscopic methods available for the characterization of the dynamics of heme protein active sites, time-resolved resonance Raman spectroscopy (TR 3) is a powerful technique because excitation within the heme p-p * electronic absorption transitions selectively enhances vibrational modes of the heme and bound-proximal/distal ligands without the interference from the modes associated with the protein matrix. On the other hand, time-resolved step-scan (TRS 2) Fourier transform infrared (FTIR) spectroscopy has the sensitivity and resolution to detect, in addition to ligands bound to metal centers and the kinetics of ligand photodissociation, transient changes at the level of individual amino acids during protein action. This review outlines the application of both TR 3 and TRS 2-FTIR to heme-copper oxidases and nitric oxide reductases.

Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba 3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved stepscan Fourier transform infrared difference spectra of photodissociated CO from heme a 3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to Cu B but is trapped within a docking site near the ring A of heme a 3 propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (CO ‫؍‬ 2131 cm ؊1 , t d ‫؍‬ 10-35 s and CO ‫؍‬ 2146 cm ؊1 , t d ‫؍‬ 85 s) are observed. At later times (t d ‫؍‬ 110 s) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herein.

We report the first study of O 2 migration in the putative O 2 channel of cytochrome ba 3 and its effect to the properties of the binuclear heme a 3-Cu B center of cytochrome ba 3 from Thermus thermophilus. The Fourier transform infrared spectra of the ba 3-CO complex demonstrate that in the presence of 60-80 M O 2 , the (C-O) of Cu B 1؉-CO at 2053 cm ؊1 (complex A) shifts to 2045 cm ؊1 and remains unchanged in H 2 O/D 2 O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the CO stretching modes of heme a 3-CO (complex B), however, remain unchanged. The change in the (C-O) of complex A results in an increase of k ؊2 , and thus in a higher affinity of Cu B for exogenous ligands. The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient Cu B 1؉-CO complex at pH 6.5 is 30.4 s ؊1 and 28.3 s ؊1 in the presence of O 2. Similarly, the rebinding to heme a 3 is slightly affected and occurs with k 2 ‫؍‬ 26.3 s ؊1 and 24.6 s ؊1 in the presence of O 2. These results provide solid evidence that in cytochrome ba 3 , the ligand delivery channel is located at the Cu B site, which is the ligand entry to the heme a 3 pocket. We suggest that the properties of the O 2 channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba 3 with O 2 and NO.

Intracavity molecular dynamics studies of photodissociated carbon monoxide from ba 3 -cytochrome c oxidase have been performed by sampling the phase space with several hundreds of trajectories each integrated up to 100 ps time interval. It is shown that the cis conformation of protonated ring-D propionate of heme a 3 and its trans conformation for the deprotonated species control the CO location by creating two distinct equilibrium states for CO confined in a cavity internal to the distal heme pocket. Thus, these cis (closed gate) and trans (open gate) conformations of heme a 3 propionate D play the role of a switch, opening or closing a gate for confining CO in a cavity internal to the heme pocket or releasing it to a bigger outer cavity. The geometry of the inner cavity and the validity of the potential function employed are further investigated by Density Functional Theory calculations for the active site, potential of mean force curves along the copper-CO bond, as well as with Quantum Mechanics/Molecular Mechanics calculations. In the light of the present study, trajectory scenarios for the dissociation of CO previously suggested from time-resolved infrared spectroscopy are re-examined.

The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O2, NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba3 from T. thermophilus, which is responsible for the activation and reduction of both O2 and NO, to gain insight into the structure of ligandbinding intermediates at ambient temperature. We show that, upon dissociation, the photolyzed CO becomes trapped within a ligand docking site located near the ring A propionate of heme a3. The 2131 cm -1 mode of the "docked" CO we have detected corresponds to the B1 state of Mb and persists for 35 µs. The release of CO from the docking site is not followed by recombination to the heme a3 Fe. Our analysis indicates that this behavior reflects a mechanism in which the protein near ring A of heme a3 propionate reorganizes about the released CO from the docking site, and establishes a transient barrier that inhibits the recombination process to the heme a3 Fe for a few milliseconds. Rebinding to heme a3 occurs with k2 ) 29.5 s -1 . These results have implications for understanding the role of ligand binding/escape through docking sites and channels in heme-copper oxidases and, thus, in respiration.

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba 3 -cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the Cu B -CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba 3 -oxidase. We assigned the bands at 507 and 568 cm ؊1 to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm ؊1 showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the ␣-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa 3oxidase, are discussed in terms of the proposed coupling of the ␣-and ␤-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The Cu B -CO complex was identified by its (CO) at 2053 cm ؊1 and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent Cu B (I) that is located at the O 2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.

We report the first study of O 2 migration in the putative O 2 channel of cytochrome ba 3 and its effect to the properties of the binuclear heme a 3 -Cu B center of cytochrome ba 3 from Thermus thermophilus. The Fourier transform infrared spectra of the ba 3 -CO complex demonstrate that in the presence of 60 -80 M O 2 , the (C-O) of Cu B 1؉ -C-O at 2053 cm ؊1 (complex A) shifts to 2045 cm ؊1 and remains unchanged in H 2 O/D 2 O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a 3 -CO (complex B), however, remain unchanged. The change in the (C-O) of complex A results in an increase of k ؊2 , and thus in a higher affinity of Cu B for exogenous ligands.

Knowledge of the structure and dynamics of
the ligand channel(s) in heme-copper oxidases is critical for
understanding how the protein environment modulates the
functions of these enzymes. Using photolabile NO and O2
carriers, we recently found that NO and O2 binding in
Thermus thermophilus (Tt ) ba3 is ∼10 times faster than in the
bovine enzyme, indicating that inherent structural differences
affect ligand access in these enzymes. Using X-ray crystallog-
raphy, time-resolved optical absorption measurements, and
theoretical calculations, we investigated ligand access in wild-
type Tt ba3 and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O2 channel of Tt ba3 are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa3 enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba3 and the T231F mutant. The results show that the Tt ba3 Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic “way station”, which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba3 and bovine aa3 show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba3 Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.