Avian cryptochrome 4 binds superoxide.

Flavin-binding cryptochromes are blue-light sensitive photoreceptors that have been implicated with magnetoreception in some species. The photocycle involves an intra-protein photo-reduction of the flavin cofactor, generating a magnetosensitive radical pair, and its subsequent re-oxidation. Superoxide (O2•-) is generated in the re-oxidation with molecular oxygen. The resulting O2•--containing radical pairs have also been hypothesised to underpin various magnetosensitive traits, but due to fast spin relaxation when tumbling in solution would require immobilisation. We here describe our insights in the binding of superoxide to cryptochrome 4 from C. livia based on extensive all-atom molecular dynamics studies and density-functional theory calculations. The positively charged "crypt" region that leads to the flavin binding pocket transiently binds O2•- at 5 flexible binding sites centred on arginine residues. Typical binding times amounted to tens of nanoseconds, but exceptional binding events extended to several hundreds of nanoseconds and slowed the rotational diffusion, thereby realising rotational correlation times as large as 1 ns. The binding sites are particularly efficient in scavenging superoxide escaping from a putative generation site close to the flavin-cofactor, possibly implying a functional relevance. We discuss our findings in view of a potential magnetosensitivity of biological flavin semiquinone/superoxide radical pairs.

Use of Gaussian-Type Functions for Describing Fast Ion-Matter Irradiation with Time-Dependent Density Functional Theory.

The electronic stopping power is an observable property that quantifies the ability of swift ions to penetrate matter to transfer energy to the electron cloud. The recent literature has proven the value of Real-Time Time-Dependent Density Functional Theory to accurately evaluate this property from first-principles, but questions remain regarding the capability of computer codes relying on atom-centered basis functions to capture the physics at play. In this Perspective, we draw attention to the fact that irradiation by swift ions triggers electron emission into the continuum, especially at the Bragg peak. We investigate the ability of Gaussian atomic orbitals (AOC), which were fitted to mimic continuum wave functions, to improve electronic stopping power predictions. AOC are added to standard correlation-consistent basis sets or STO minimal basis sets. Our benchmarks for water irradiation by fast protons clearly advocate for the use of AOC, especially near the Bragg peak. We show that AOC only need to be placed on the molecules struck by the ion. The number of AOC that are added to the usual basis set is relatively small compared to the total number of atomic orbitals, making the use of such a basis set an excellent choice from a computational cost point of view. The optimum basis set combination is applied for the calculation of the stopping power of a proton in water with encouraging agreement with experimental data.

Irradiation of Plutonium Tributyl Phosphate Complexes by Ionizing Alpha Particles: A Computational Study.

The PUREX solvent extraction process, widely used for recovering uranium and plutonium from spent nuclear fuel, utilizes an organic solvent composed of tributyl phosphate (TBP). The emission of ionizing particles such as alpha particles, resulting from the decay of plutonium, makes the organic solvent vulnerable to degradation. Here, we study the ultrashort time alpha irradiation of tributylphosphate (TBP) and Pu(NO3)4(TBP)2 complex formed in the PUREX process. Electron dynamics is propagated by Real-Time-Dependent Auxiliary Density Functional Theory (RT-TD-ADFT). We investigate the use of previously proposed absorption boundary conditions (ABC) in the molecular orbital space to treat secondary electron emission. Basis set and exchange correlation functional effects with ABC are reported as well as a detailed analysis of the ABC parametrization. Preliminary results on the water molecule and then on TBP show that the phenomenological nature of the ABC parameters necessitates selecting appropriate values for each system under study. Irradiation of free and complexed TBP shows an influence of the ligands on the variation of atomic charges on the femtosecond time scale. An accumulation of atomic charges in the alkyl chains of TBP is observed in the case where the nitrate groups are predominantly irradiated. In addition, we find that the Pu atom regains its electric charge very rapidly after being hit by the projectile, with the coordination sphere serving as an electron reservoir to preserve its formal redox state. This study paves the road toward a full understanding of the degradation of organic extracants employed in the nuclear industry.

On the Role of Charge Transfer in Many-Body Non-Covalent Interactions.

Charge transfer is one of the mechanisms involved in non-covalent interactions. In molecular dimers, its contribution to pairwise interaction energies has been studied extensively using a variety of interaction energy decomposition schemes. In polar interactions such as hydrogen bonds, it can contribute ten or several tens of percent of the interaction energy. Less is known about its importance in higher-order interactions in many-body systems, mainly because of the lack of methods applicable to this problem. In this work, we extend our method for the quantification of the charge-transfer energy based on constrained DFT to many-body cases and apply it to model trimers extracted from molecular crystals. Our calculations show that charge transfer can account for a large fraction of the total three-body interaction energy. This also has implications for DFT calculations of many-body interactions in general as it is known that many DFT functionals struggle to describe charge-transfer effects correctly.

Ab initio derivation of flavin hyperfine interactions for the protein magnetosensor cryptochrome.

The radicals derived from flavin adenine dinucleotide (FAD) are a corner stone of recent hypotheses about magnetoreception, including the compass of migratory songbirds. These models attribute a magnetic sense to coherent spin dynamics in radical pairs within the flavo-protein cryptochrome. The primary determinant of sensitivity and directionality of this process are the hyperfine interactions of the involved radicals. Here, we present a comprehensive computational study of the hyperfine couplings in the protonated and unprotonated FAD radicals in cryptochrome 4 from C. livia. We combine long (800 ns) molecular dynamics trajectories to accurate quantum chemistry calculations. Hyperfine parameters are derived using auxiliary density functional theory applied to cluster and hybrid QM/MM (Quantum Mechanics/Molecular Mechanics) models comprising the FAD and its significant surrounding environment, as determined by a detailed sensitivity analysis. Thanks to this protocol we elucidate the sensitivity of the hyperfine interaction parameters to structural fluctuations and the polarisation effect of the protein environment. We find that the ensemble-averaged hyperfine interactions are predominantly governed by thermally induced geometric distortions of the flavin. We discuss our results in view of the expected performance of these radicals as part of a magnetoreceptor. Our data could be used to parametrize spin Hamiltonians including not only average values but also standard deviations.

Following the density evolution using real time density functional theory and density based indexes: Application to model push-pull molecules.

Considering as test case a family of organic rod like push-pull molecules, we derived and applied density based index enabling the description and diagnostic of the electronic density evolution in real time-time dependent density functional theory (RT-TDDFT) simulations. In particular, both the charge transfer (CT) distance and a diagnostic index, the DCT and MAC RT respectively, were computed on the fly from the density distribution obtained at a given time and the reference ground state density and their mean values were compared with what obtained at Linear Response-TDDFT level. Besides giving a way of analyzing the density redistribution occurring in time, these tools allowed to show how RT-TDDFT, which is definitely a powerful method to model the evolution of the density in CT or charge separation processes, can be affected by the same artifacts known for LR-TDDFT approaches and, particularly, to those related to the use of approximate exchange correlation functionals. The analysis here performed allowed to identify and discard on fly the electronic configurations corresponding to spurious situations.

Insights into the effect of distal histidine and water hydrogen bonding on NO ligation to ferrous and ferric heme: a DFT study.

The effect of distal histidine on ligation of NO to ferrous and ferric-heme, has been investigated with the high-level density functional theoretical (DFT) method. It has been predicted that the distal histidine significantly stabilizes the interaction of NO ferrous-heme (by -2.70 kcal mol-1). Also, water hydrogen bonding is quite effective in strengthening the Fe-NO bond in ferrous heme. In contrast in ferric heme, due to the large distance between the H2O and O(NO) and lack of hydrogen bonding, the distal histidine exhibits only a slight effect on the binding of NO to the ferric analogue. Concerning the bond nature of FeII-NO and FeIII-NO in heme, a QTAIM analysis predicts a partially covalent and ionic bond nature in both systems.

Anisotropic magnetic field effects in the re-oxidation of cryptochrome in the presence of scavenger radicals.

The avian compass and many other of nature's magnetoreceptive traits are widely ascribed to the protein cryptochrome. There, magnetosensitivity is thought to emerge as the spin dynamics of radicals in the applied magnetic field enters in competition with their recombination. The first and dominant model makes use of a radical pair. However, recent studies have suggested that magnetosensitivity could be markedly enhanced for a radical triad, the primary radical pair of which undergoes a spin-selective recombination reaction with a third radical. Here, we test the practicality of this supposition for the reoxidation reaction of the reduced FAD cofactor in cryptochrome, which has been implicated with light-independent magnetoreception but appears irreconcilable with the classical radical pair mechanism (RPM). Based on the available realistic cryptochrome structures, we predict the magnetosensitivity of radical triad systems comprising the flavin semiquinone, the superoxide, and a tyrosine or ascorbyl scavenger radical. We consider many hyperfine-coupled nuclear spins, the relative orientation and placement of the radicals, their coupling by the electron-electron dipolar interaction, and spin relaxation in the superoxide radical in the limit of instantaneous decoherence, which have not been comprehensively considered before. We demonstrate that these systems can provide superior magnetosensitivity under realistic conditions, with implications for dark-state cryptochrome magnetoreception and other biological magneto- and isotope-sensitive radical recombination reactions.

The mystery of sub-picosecond charge transfer following irradiation of hydrated uridine monophosphate.

The early mechanisms by which ionizing rays damage biological structures by so-called direct effects are largely elusive. In a recent picosecond pulse radiolysis study of concentrated uridine monophosphate solutions [J. Ma, S. A. Denisov, J.-L. Marignier, P. Pernot, A. Adhikary, S. Seki and M. Mostafavi, J. Phys. Chem. Lett., 2018, 9, 5105], unexpected results were found regarding the oxidation of the nucleobase. The signature of the oxidized nucleobase could not be detected 5 ps after the electron pulse, but only the oxidized phosphate, raising intriguing questions about the identity of charge-transfer mechanisms that could explain the absence of U+. We address here this question by means of advanced first-principles atomistic simulations of solvated uridine monophosphate, combining Density Functional Theory (DFT) with polarizable embedding schemes. We contrast three very distinct mechanisms of charge transfer covering the atto-, femto- and pico-second timescales. We first investigate the ionization mechanism and subsequent hole/charge migrations on a timescale of attoseconds to a few femtoseconds under the frozen nuclei approximation. We then consider a nuclear-driven phosphate-to-oxidized-nucleobase electron transfer, showing that it is an uncompetitive reaction channel on the sub-picosecond timescale, despite its high exothermicity and significant electronic coupling. Finally, we show that non-adiabatic charge transfer is enabled by femtosecond nuclear relaxation after ionization. We show that electronic decoherence and the electronic coupling strength are the key parameters that determine the hopping probabilities. Our results provide important insight into the interplay between electronics and nuclear motions in the early stages of the multiscale responses of biological matter subjected to ionizing radiation.

Mechanistic Insights on Heme-to-Heme Transmembrane Electron Transfer Within NADPH Oxydases From Atomistic Simulations.

NOX5 is a member of the NADPH oxidase family which is dedicated to the production of reactive oxygen species. The molecular mechanisms governing transmembrane electron transfer (ET) that permits to shuttle electrons over the biological membrane have remained elusive for a long time. Using computer simulations, we report conformational dynamics of NOX5 embedded within a realistic membrane environment. We assess the stability of the protein within the membrane and monitor the existence of cavities that could accommodate dioxygen molecules. We investigate the heme-to-heme electron transfer. We find a reaction free energy of a few tenths of eV (ca. -0.3 eV) and a reorganization free energy of around 1.1 eV (0.8 eV after including electrostatic induction corrections). The former indicates thermodynamically favorable ET, while the latter falls in the expected values for transmembrane inter-heme ET. We estimate the electronic coupling to fall in the range of the µeV. We identify electron tunneling pathways showing that not only the W378 residue is playing a central role, but also F348. Finally, we reveal the existence of two connected O2-binding pockets near the outer heme with fast exchange between the two sites on the nanosecond timescale. We show that when the terminal heme is reduced, O2 binds closer to it, affording a more efficient tunneling pathway than when the terminal heme is oxidized, thereby providing an efficient mechanism to catalyze superoxide production in the final step. Overall, our study reveals some key molecular mechanisms permitting reactive oxygen species production by NOX5 and paves the road for further investigation of ET processes in the wide family of NADPH oxidases by computer simulations.

First-Principles Simulations of Biological Molecules Subjected to Ionizing Radiation.

Ionizing rays cause damage to genomes, proteins, and signaling pathways that normally regulate cell activity, with harmful consequences such as accelerated aging, tumors, and cancers but also with beneficial effects in the context of radiotherapies. While the great pace of research in the twentieth century led to the identification of the molecular mechanisms for chemical lesions on the building blocks of biomacromolecules, the last two decades have brought renewed questions, for example, regarding the formation of clustered damage or the rich chemistry involving the secondary electrons produced by radiolysis. Radiation chemistry is now meeting attosecond science, providing extraordinary opportunities to unravel the very first stages of biological matter radiolysis. This review provides an overview of the recent progress made in this direction, focusing mainly on the atto- to femto- to picosecond timescales. We review promising applications of time-dependent density functional theory in this context.

The physical stage of radiolysis of solvated DNA by high-energy-transfer particles: insights from new first principles simulations.

The primary processes that occur following direct irradiation of bio-macromolecules by ionizing radiation determine the multiscale responses that lead to biomolecular lesions. The so-called physical stage loosely describes processes of energy deposition and molecular ionization/excitation but remains largely elusive. We propose a new approach based on first principles density functional theory to simulate energy deposition in large and heterogeneous biomolecules by high-energy-transfer particles. Unlike traditional Monte Carlo approaches, our methodology does not rely on pre-parametrized sets of cross-sections, but captures excitation, ionization and low energy electron emission at the heart of complex biostructures. It furthermore gives access to valuable insights on ultrafast charge and hole dynamics on the femtosecond time scale. With this new tool, we reveal the mechanisms of ionization by swift ions in microscopic DNA models and solvated DNA comprising almost 750 atoms treated at the DFT level of description. We reveal a so-called ebb-and-flow ionization mechanism in which polarization of the irradiated moieties appears as a key feature. We also investigate where secondary electrons produced by irradiation localize on chemical moieties composing DNA. We compare irradiation of solvated DNA by light (H+, and He2+) vs. heavier (C6+) ions, highlighting the much higher probability of double ionization with the latter. Our methodology constitutes a stepping stone towards a greater understanding of the chemical stage and more generally towards the multiscale modelling of radiation damage in biology using first principles.

Water binding to FeIII hemes studied in a cooled ion trap: characterization of a strong 'weak' ligand.

The interaction of a water molecule with ferric heme-iron protoporphyrin ([PP FeIII]+) has been investigated in the gas phase in an ion trap and studied theoretically by density functional theory. It is found that the interaction of water with ferric heme leads to a stable [PP-FeIII-H2O]+ complex in the intermediate spin state (S = 3/2), in the same state as its unligated [PP-FeIII]+ homologue, without spin crossing during water attachment. Using the Van't Hoff equation, the reaction enthalpy for the formation of a Fe-OH2 bond has been determined for [PP-FeIII-H2O]+ and [PP-FeIII-(H2O)2]+. The corrected binding energy for a single Fe-H2O bond is -12.2 ± 0.6 kcal mol-1, while DFT calculations at the OPBE level yield -11.7 kcal mol-1. The binding energy of the second ligation yielding a six coordinated FeIII atom is decreased with a bond energy of -9 ± 0.9 kcal mol-1, well reproduced by calculations as -7.1 kcal mol-1. However, calculations reveal features of a weaker bond type, such as a rather long Fe-O bond with 2.28 Å for the [PP-FeIII-H2O]+ complex and the absence of a spin change by complexation. Thus despite a strong bond with H2O, the FeIII atom does not show, through theoretical modelling, a strong acceptor character in its half filled 3dz2 orbital. It is also observed that the binding properties of H2O to hemes seem strikingly specific to ferric heme and we have shown, experimentally and theoretically, that the affinity of H2O for protonated heme [H PP-Fe]+, an intermediate between FeIII and FeII, is strongly reduced compared to that for ferric heme.

Molecular Simulations with in-deMon2k QM/MM, a Tutorial-Review.

deMon2k is a readily available program specialized in Density Functional Theory (DFT) simulations within the framework of Auxiliary DFT. This article is intended as a tutorial-review of the capabilities of the program for molecular simulations involving ground and excited electronic states. The program implements an additive QM/MM (quantum mechanics/molecular mechanics) module relying either on non-polarizable or polarizable force fields. QM/MM methodologies available in deMon2k include ground-state geometry optimizations, ground-state Born-Oppenheimer molecular dynamics simulations, Ehrenfest non-adiabatic molecular dynamics simulations, and attosecond electron dynamics. In addition several electric and magnetic properties can be computed with QM/MM. We review the framework implemented in the program, including the most recently implemented options (link atoms, implicit continuum for remote environments, metadynamics, etc.), together with six applicative examples. The applications involve (i) a reactivity study of a cyclic organic molecule in water; (ii) the establishment of free-energy profiles for nucleophilic-substitution reactions by the umbrella sampling method; (iii) the construction of two-dimensional free energy maps by metadynamics simulations; (iv) the simulation of UV-visible absorption spectra of a solvated chromophore molecule; (v) the simulation of a free energy profile for an electron transfer reaction within Marcus theory; and (vi) the simulation of fragmentation of a peptide after collision with a high-energy proton.

Multicomponent density functional theory with density fitting.

Multicomponent Density Functional Theory (MDFT) is a promising methodology to incorporate nuclear quantum effects, such as zero-point energy or tunneling, or to simulate other types of particles such as muons or positrons using particle densities as basic quantities. As for standard electronic DFT, a still ongoing challenge is to achieve the most efficient implementations. We introduce a multicomponent DFT implementation within the framework of auxiliary DFT, focusing on molecular systems comprising electrons and quantum protons. We introduce a dual variational procedure to determine auxiliary electron and proton densities which leads to a succession of approximate energy expressions. Electronic and protonic fitted densities are employed in (i) electron-electron, proton-proton, and electron-proton classical Coulomb interactions and (ii) electron exchange-correlation, proton-proton exchange, and electron-proton correlation (EPC) potentials. If needed, exact exchange among electrons or among protons is computed by the variational fitting of the corresponding Fock potential. The implementation is carried out in deMon2k. We test various electron proton correlation functionals on proton affinities. We find that auxiliary densities can be safely used in electron-electron, proton-proton, and electron-proton classical Coulomb interactions, as well as in EPC, albeit with some precautions related to the choice of the electronic auxiliary basis set that must be flexible enough. Computational tests reported indicate that introduction of density fitting in MDFT is clearly advantageous in terms of computational effort with good scaling properties with respect to the number of electrons and protons treated at the DFT level.

The dramatic effect of N-methylimidazole on trans axial ligand binding to ferric heme: experiment and theory.

The binding energy of CO, O2 and NO to isolated ferric heme, [FeIIIP]+, was studied in the presence and absence of a σ donor (N-methylimidazole and histidine) as the trans axial ligand. This study combines the experimental determination of binding enthalpies by equilibrium measurements in a low temperature ion trap using the van't Hoff equation and high level DFT calculations. It was found that the presence of N-methylimidazole as the axial ligand on the [FeIIIP]+ porphyrin dramatically weakens the [FeIIIP-ligand]+ bond with an up to sevenfold decrease in binding energy owing to the σ donation by N-methylimidazole to the FeIII(3d) orbitals. This trans σ donor effect is characteristic of ligation to iron in hemes in both ferrous and ferric redox forms; however, to date, this has not been observed for ferric heme.

Gating the electron transfer at a monocopper centre through the supramolecular coordination of water molecules within a protein chamber mimic.

Functionality of enzymes is strongly related to water dynamic processes. The control of the redox potential for metallo-enzymes is intimately linked to the mediation of water molecules in the first and second coordination spheres. Here, we report a unique example of supramolecular control of the redox properties of a biomimetic monocopper complex by water molecules. It is shown that the copper complex based on a calix[6]arene covalently capped with a tetradentate [tris(2-methylpyridyl)amine] (tmpa) core, embedding the metal ion in a hydrophobic cavity, can exist in three different states. The first system displays a totally irreversible redox behaviour. It corresponds to the reduction of the 5-coordinate mono-aqua-CuII complex, which is the thermodynamic species in the +II state. The second system is detected at a high redox potential. It is ascribed to an "empty cavity" or "water-free" state, where the CuI ion sits in a 4-coordinate trigonal environment provided by the tmpa cap. This complex is the thermodynamic species in the +I state under "dry conditions". Surprisingly, a third redox system appears as the water concentration is increased. Under water-saturation conditions, it displays a pseudo-reversible behaviour at a low scan rate at the mid-point from the water-free and aqua species. This third system is not observed with the Cu-tmpa complex deprived of a cavity. In the calix[6]cavity environment, it is ascribed to a species where a pair of water molecules is hosted by the calixarene cavity. A molecular mechanism for the CuII/CuI redox process with an interplay of (H2O) x (x = 0, 1, 2) hosting is proposed on the basis of computational studies. Such an unusual behaviour is ascribed to the unexpected stabilization of the CuI state by inclusion of the pair of water molecules. This phenomenon strongly evidences the drastic influence of the interaction between water molecules and a hydrophobic cavity on controlling the thermodynamics and kinetics of the CuII/CuI electron transfer process.

The surprisingly high ligation energy of CO to ruthenium porphyrins.

A combined theoretical and experimental approach has been used to investigate the binding energy of a ruthenium metalloporphyrin ligated with CO, ruthenium tetraphenylporphyrin [RuII TPP], in the RuII oxidation degree. Measurements performed with VUV ionization using the DESIRS beamline at Synchrotron SOLEIL led to adiabatic ionization energies of [RuII TPP] and its complex with CO, [RuII TPP-CO], of 6.48 ± 0.03 eV and 6.60 ± 0.03 eV, respectively, while the ion dissociation threshold of [RuII TPP-CO]+ is measured to be 8.36 ± 0.03 eV using the ground-state neutral complex. These experimental data are used to derive the binding energies of the CO ligand in neutral and cationic complexes (1.88 ± 0.06 eV and 1.76 ± 0.06 eV, respectively) using a Born-Haber cycle. Density functional theory calculations, in very satisfactory agreement with the experimental results, help to get insights into the metal-ligand bond. Notably, the high ligation energies can be rationalized in terms of the ruthenium orbital structure, which is singular compared to that of the iron atom. Thus, beyond indications of a strengthening of the Ru-CO bond due to the decrease in the CO vibrational frequency in the complex as compared to the Fe-CO bond, high-level calculations are essential to accurately describe the metal ligand (CO) bond and show that the Ru-CO bond energy is strongly affected by the splitting of triplet and singlet spin states in uncomplexed [Ru TPP].

AMOEBA Polarizable Force Field Parameters of the Heme Cofactor in Its Ferrous and Ferric Forms.

We report the first parameters of the heme redox cofactors for the polarizable AMOEBA force field in both the ferric and ferrous forms. We consider two types of complexes, one with two histidine side chains as axial ligands and one with a histidine and a methionine side chain as ligands. We have derived permanent multipoles from second-order Møller-Plesset perturbation theory (MP2). The sets of parameters have been validated in a first step by comparison of AMOEBA interaction energies of heme and a collection of biologically relevant molecules with MP2 and Density Functional Theory (DFT) calculations. In a second validation step, we consider interaction energies with large aggregates comprising around 80 H2O molecules. These calculations are repeated for 30 structures extracted from semiempirical PM7 DM simulations. Very encouraging agreement is found between DFT and the AMOEBA force field, which results from an accurate treatment of electrostatic interactions. We finally report long (10 ns) MD simulations of cytochromes in two redox states with AMOEBA testing both the 2003 and 2014 AMOEBA water models. These simulations have been carried out with the TINKER-HP (High Performance) program. In conclusion, owing to their ubiquity in biology, we think the present work opens a wide array of applications of the polarizable AMOEBA force field on hemeproteins.