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2 H solid-state NMR and atomistic molecular dynamics (MD) simulations are used to understand the disorder of guest solvent molecules in two cocrystal solvates of the pharmaceutical furosemide. Traditional approaches to interpreting the NMR data fail to provide a coherent model of molecular behavior and indeed give misleading kinetic data. In contrast, the direct prediction of the NMR properties from MD simulation trajectories allows the NMR data to be correctly interpreted in terms of combined jump-type and libration-type motions. Time-independent component analysis of the MD trajectories provides additional insights, particularly for motions that are invisible to NMR. This allows a coherent picture of the dynamics of molecules restricted in molecular-sized cavities to be determined.

Organic thermoelectric materials have potential for wearable heating, cooling, and energy generation devices at room temperature. For this to be technologically viable, high-conductance ( G ) and high-Seebeck-coefficient ( S ) materials are needed. For most semiconductors, the increase in S is accompanied by a decrease in G . Here, using a combined experimental and theoretical investigation, we demonstrate that a simultaneous enhancement of S and G can be achieved in single organic radical molecules, thanks to their intrinsic spin state. A counterintuitive quantum interference (QI) effect is also observed in stable Blatter radical molecules, where constructive QI occurs for a meta -connected radical, leading to further enhancement of thermoelectric properties. Compared to an analogous closed-shell molecule, the power factor is enhanced by more than 1 order of magnitude in radicals. These results open a new avenue for the development of organic thermoelectric materials operating at room temperature.

In the study of biological structures, pulse dipolar spectroscopy (PDS) is used to elucidate spin-spin distances at nanometre-scale by measuring dipole-dipole interactions between paramagnetic centres. The PDS methods of Double Electron Electron Resonance (DEER) and Relaxation Induced Dipolar Modulation Enhancement (RIDME) are employed, and their results compared, for the measurement of the dipolar coupling between nitroxide spin labels and copper-II (Cu(II)) paramagnetic centres within the copper amine oxidase from Arthrobacter globiformis (AGAO). The distance distribution results obtained indicate that two distinct distances can be measured, with the longer of these at c.a. 5 nm. Conditions for optimising the RIDME experiment such that it may outperform DEER for these long distances are discussed. Modelling methods are used to show that the distances obtained after data analysis are consistent with the structure of AGAO., Supplementary Information: The online version contains supplementary material available at 10.1007/s00723-021-01321-6., (© The Author(s) 2021.)

SHIP2 (SH2-domain containing inositol 5-phosphatase type 2) is a canonical 5-phosphatase, which, through its catalytic action on PtdInsP 3 , regulates the PI3K/Akt pathway and metabolic action of insulin. It is a drug target, but there is limited evidence of inhibition of SHIP2 by small molecules in the literature. With the goal to investigate inhibition, we report a homologous family of synthetic, chromophoric benzene phosphate substrates of SHIP2 that display the headgroup regiochemical hallmarks of the physiological inositide substrates that have proved difficult to crystallize with 5-phosphatases. Using time-dependent density functional theory (TD-DFT), we explore the intrinsic fluorescence of these novel substrates and show how fluorescence can be used to assay enzyme activity. The TD-DFT approach promises to inform rational design of enhanced active site probes for the broadest family of inositide-binding/metabolizing proteins, while maintaining the regiochemical properties of bona fide inositide substrates., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Two new cobalt bis-iminopyridines, [Co(DDP)(H2O)2](NO3)2 (1, DDP = cis-[1,3-bis(2-pyridinylenamine)] cyclohexane) and [Co(cis-DDOP)(NO3)](NO3) (2, cis-DDOP = cis-3,5-bis[(2-Pyridinyleneamin]-trans-hydroxycyclohexane) electrocatalyse the 4-proton, 4-electron reduction of acetonitrile to ethylamine. For 1, this reduction occurs in preference to reduction of protons to H2. A coordinating hydroxyl proton relay in 2 reduces the yield of ethylamine and biases the catalytic system back towards H2.

Of the many biophysical techniques now being brought to bear on studies of membranes, electron paramagnetic resonance (EPR) of nitroxide spin probes was the first to provide information about both mobility and ordering in lipid membranes. Here, we report the first prediction of variable temperature EPR spectra of model lipid bilayers in the presence and absence of cholesterol from the results of large scale fully atomistic molecular dynamics (MD) simulations. Three types of structurally different spin probes were employed in order to study different parts of the bilayer. Our results demonstrate very good agreement with experiment and thus confirm the accuracy of the latest lipid force fields. The atomic resolution of the simulations allows the interpretation of the molecular motions and interactions in terms of their impact on the sensitive EPR line shapes. Direct versus indirect effects of cholesterol on the dynamics of spin probes are analysed. Given the complexity of structural organisation in lipid bilayers, the advantage of using a combined MD-EPR simulation approach is two-fold. Firstly, prediction of EPR line shapes directly from MD trajectories of actual phospholipid structures allows unambiguous interpretation of EPR spectra of biological membranes in terms of complex motions. Secondly, such an approach provides an ultimate test bed for the up-to-date MD simulation models employed in the studies of biological membranes, an area that currently attracts great attention., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We report the first application of fully atomistic molecular dynamics (MD) simulations to the prediction of electron paramagnetic resonance (EPR) spectra of spin labelled DNA. Models for two structurally different DNA spin probes with either the rigid or flexible position of the nitroxide group in the base pair, employed in experimental studies previously, have been developed. By the application of the combined MD-EPR simulation methodology we aimed at the following. Firstly, to provide a test bed against a sensitive spectroscopic technique for the recently developed improved version of the parmbsc1 force field for MD modelling of DNA. The predicted EPR spectra show good agreement with the experimental ones available from the literature, thus confirming the accuracy of the currently employed DNA force fields. Secondly, to provide a quantitative interpretation of the motional contributions into the dynamics of spin probes in both duplex and single-strand DNA fragments and to analyse their perturbing effects on the local DNA structure. Finally, a combination of MD and EPR allowed us to test the validity of the application of the Model-Free (M-F) approach coupled with the partial averaging of magnetic tensors to the simulation of EPR spectra of DNA systems by comparing the resultant EPR spectra with those simulated directly from MD trajectories. The advantage of the M-F based EPR simulation approach over the direct propagation techniques is that it requires motional and order parameters that can be calculated from shorter MD trajectories. The reported MD-EPR methodology is transferable to the prediction and interpretation of EPR spectra of higher order DNA structures with novel types of spin labels.

Antimicrobial peptides (AMPs) are small cationic proteins that are able to destabilize a lipid bilayer structure through one or more modes of action. In this study, we investigate the processes of peptide aggregation and pore formation in lipid bilayers and vesicles by the highly cationic AMP, Chrysophsin-3 (chrys-3), using coarse-grained molecular dynamics (CG-MD) simulations and potential of mean force calculations. We study long 50 μs simulations of chrys-3 at different concentrations, both at the surface of dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylcholine (POPC) bilayers, and also interacting within the interior of the lipid membrane. We show that aggregation of peptides at the surface, leads to pronounced deformation of lipid bilayers, leading in turn to lipid protrusions for peptide : ligand ratios > 1 : 12. In addition, aggregation of chrys-3 peptides within the centre of a lipid bilayer leads to spontaneous formation of pores and aggregates. Both mechanisms of interaction are consistent with previously reported experimental data for chrys-3. Similar results are observed also in POPC vesicles and mixed lipid bilayers composed of the zwitterionic lipid palmitoyloleoylphosphatidylethanolamine (POPE) and the negatively charged lipid palmitoyloleoylphosphatidylglycerol (POPG). The latter are employed as models of the bacterial membrane of Escherichia coli.

We report the first application of fully atomistic molecular dynamics (MD) simulations to the prediction of the motional electron paramagnetic resonance (EPR) spectra of lyotropic liquid crystals in different aggregation states doped with a paramagnetic spin probe. The purpose of this study is twofold. First, given that EPR spectra are highly sensitive to the motions and order of the spin probes doped within lyotropic aggregates, simulation of EPR line shapes from the results of MD modelling provides an ultimate test bed for the force fields currently employed to model such systems. Second, the EPR line shapes are simulated using the motional parameters extracted from MD trajectories using the Model-Free (MF) approach. Thus a combined MD-EPR methodology allowed us to test directly the validity of the application of the MF approach to systems with multi-component molecular motions. All-atom MD simulations using the General AMBER Force Field (GAFF) have been performed on sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium chloride (DTAC) liquid crystals. The resulting MD trajectories were used to predict and interpret the EPR spectra of pre-micellar, micellar, rod and lamellar aggregates. The predicted EPR spectra demonstrate good agreement with most of experimental line shapes thus confirming the validity of both the force fields employed and the MF approach for the studied systems. At the same time simulation results confirm that GAFF tends to overestimate the packing and the order of the carbonyl chains of the surfactant molecules., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

EPR studies combined with fully atomistic Molecular Dynamics (MD) simulations and an MD-EPR simulation method provide evidence for intrinsic low rotameric mobility of a nitroxyl spin label, Rn, compared to the more widely employed label MTSL (R1). Both experimental and modelling results using two structurally different sites of attachment to Myoglobin show that the EPR spectra of Rn are more sensitive to the local protein environment than that of MTSL. This study reveals the potential of using the Rn spin label as a reporter of protein motions., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Despite the vast amount of experimental and theoretical studies on the binding affinity of cations - especially the biologically relevant Na + and Ca 2+ - for phospholipid bilayers, there is no consensus in the literature. Here we show that by interpreting changes in the choline headgroup order parameters according to the 'molecular electrometer' concept [Seelig et al., Biochemistry, 1987, 26, 7535], one can directly compare the ion binding affinities between simulations and experiments. Our findings strongly support the view that in contrast to Ca 2+ and other multivalent ions, Na + and other monovalent ions (except Li + ) do not specifically bind to phosphatidylcholine lipid bilayers at sub-molar concentrations. However, the Na + binding affinity was overestimated by several molecular dynamics simulation models, resulting in artificially positively charged bilayers and exaggerated structural effects in the lipid headgroups. While qualitatively correct headgroup order parameter response was observed with Ca 2+ binding in all the tested models, no model had sufficient quantitative accuracy to interpret the Ca 2+ :lipid stoichiometry or the induced atomistic resolution structural changes. All scientific contributions to this open collaboration work were made publicly, using nmrlipids.blogspot.fi as the main communication platform.

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe 2 (NO) 4 (Cys) 2 ]) and Roussin's Black Salt (RBS, [Fe 4 (NO) 7 S 3 ]. In the latter case, the absence of 32 S/ 34 S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The chemistry of metal hydrides is implicated in a range of catalytic processes at metal centers. Gaining insight into the formation of such sites by protonation and/or electronation is therefore of significant value in fully exploiting the potential of such systems. Here, we show that the muonium radical (Mu . ), used as a low isotopic mass analogue of hydrogen, can be exploited to probe the early stages of hydride formation at metal centers. Mu . undergoes the same chemical reactions as H . and can be directly observed due to its short lifetime (in the microseconds) and unique breakdown signature. By implanting Mu . into three models of the [FeFe]-hydrogenase active site we have been able to detect key muoniated intermediates of direct relevance to the hydride chemistry of these systems., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Three structural isomers of tris{bis(trifluoromethyl)phenyl}borane have been studied as the acidic component of frustrated Lewis pairs. While the 3,5-substituted isomer is already known to heterolytically cleave H2 to generate a bridging-hydride; ortho-substituents in the 2,4- and 2,5-isomers quench such reactivity through electron donation into the vacant boron pz orbital and steric blocking of the boron centre; as shown by electrochemical, structural and computational studies. Electrochemical studies of the corresponding borohydrides identify that the two-electron oxidation of terminal-hydrides occurs at more positive potentials than observed for [HB(C6F5)3](-), while the bridging-hydride oxidizes at a higher potential still, comparable to that of free H2.

Reaction of [IrCp*Cl2 ]2 with ferrocenylimines (Fc=NAr, Ar=Ph, p-MeOC6 H4 ) results in ferrocene C-H activation and the diastereoselective synthesis of half-sandwich iridacycles of relative configuration Sp *,RIr *. Extension to (S)-2-ferrocenyl-4-(1-methylethyl)oxazoline gave highly diastereoselective control over the new elements of planar chirality and metal-based pseudo-tetrahedral chirality, to give both neutral and cationic half-sandwich iridacycles of absolute configuration Sc ,Sp ,RIr . Substitution reactions proceed with retention of configuration, with the planar chirality controlling the metal-centred chirality through an iron-iridium interaction in the coordinatively unsaturated cationic intermediate., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

EPR spectroscopy and theoretical data show that the slow heterogeneous electron-transfer kinetics associated with the reduction of an 18-electron Mo(IV) acetato dihydride are a consequence of an η(2)-η(1) rearrangement of the carboxylate ligand which gives a unique paramagnetic 17-electron Mo(III) dihydride.

The transfer rate of a probe molecule across the interfacial layer of a water-in-oil (w/o) microemulsion was investigated using a combination of transverse field muon spin rotation (TF-μSR), avoided level crossing muon spin resonance (ALC-μSR), and Monte Carlo simulations. Reverse microemulsions consist of nanometer-sized water droplets dispersed in an apolar solvent separated by a surfactant monolayer. Although the thermodynamic, static model of these systems has been well described, our understanding of their dynamics is currently incomplete. For example, what is the rate of solute transfer between the aqueous and apolar solvents, and how this is influenced by the structure of the interface? With an appropriate choice of system and probe molecule, μSR offers a unique opportunity to directly probe these interfacial transfer dynamics. Here, we have employed a well characterized w/o microemulsion stabilized by bis(2-ethylhexyl) sodium sulfosuccinate (Aerosol OT), with allyl alcohol (CH2═CH-CH2-OH, AA) as the probe. Resonances due to both muoniated radicals, CMuH2-C*H-CH2-OH and C*H2-CHMu-CH2-OH, were observed with the former being the dominant species. All resonances displayed solvent dependence, with those in the microemulsion observed as a single resonance located at intermediate magnetic fields to those present in either of the pure solvents. Observation of a single resonance is strong evidence for interfacial transfer being in the fast exchange limit. Monte Carlo calculations of the ΔM = 0 ALC resonances are consistent with the experimental data, indicating exchange rates greater than 10(9) s(-1), placing the rate of interfacial transfer at the diffusion limit.

Frustrated Lewis pairs have found many applications in the heterolytic activation of H2 and subsequent hydrogenation of small molecules through delivery of the resulting proton and hydride equivalents. Herein, we describe how H2 can be preactivated using classical frustrated Lewis pair chemistry and combined with in situ nonaqueous electrochemical oxidation of the resulting borohydride. Our approach allows hydrogen to be cleanly converted into two protons and two electrons in situ, and reduces the potential (the required energetic driving force) for nonaqueous H2 oxidation by 610 mV (117.7 kJ mol(-1)). This significant energy reduction opens routes to the development of nonaqueous hydrogen energy technology.

We report a general approach for the simulation of the electron paramagnetic resonance (EPR) spectra of spin labels attached to peptides and proteins directly from replica-exchange molecular dynamics (REMD) trajectories. Conventional MD trajectories are generally inadequate for the prediction of EPR line shapes since the label can become trapped in one or more of a set of rotameric states, thus preventing both conformational sampling and accurate estimates of the exchange rates between different rotamers. The advantage of using REMD is its ability to provide both efficient conformational sampling and kinetic information for spin-label dynamics. Our approach is illustrated with spin-labeled peptide. This approach to REMD-EPR simulation paves the way for the wider application of MD modeling to the simulation and interpretation of EPR spectra of spin-labeled molecules.

Bacterial NOR (nitric oxide reductase) is a major source of the powerful greenhouse gas N2O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b3 in close proximity with non-haem iron, FeB. In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between FeIII haem b3 and FeBIII (both S=5/2). On the basis of resonance Raman studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122, 9344-9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J=-1.7 cm-1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.

We report a kinetic and mechanistic study into the one-electron reduction of the archetypal Lewis acid tris(pentafluorophenyl)borane, B(C(6)F(5))(3), in dichloromethane and 1,2-difluorobenzene. Electrochemical experiments, combined with digital simulations, DFT computational studies and multinuclear NMR analysis allow us to obtain thermodynamic, kinetic and mechanistic information relating to the redox activity of B(C(6)F(5))(3). We show that tris(pentafluorophenyl)borane undergoes a quasi-reversible one-electron reduction followed by rapid chemical decomposition of the B(C(6)F(5))(3)˙(-) radical anion intermediate via a solvolytic radical pathway. The reaction products form various four-coordinate borates of which [B(C(6)F(5))(4)](-) is a very minor product. The rate of the follow-up chemical step has a pseudo-first order rate constant of the order of 6 s(-1). This value is three orders of magnitude larger than that found in previous studies performed in the donor solvent, tetrahydrofuran. The standard reduction potential of B(C(6)F(5))(3) is reported for the first time as -1.79 ± 0.1 V and -1.65 ± 0.1 V vs. ferrocene/ferrocenium in dichloromethane and 1,2-difluorobenzene respectively.

Paramagnetic hydrides are likely intermediates in hydrogen-evolving enzymic and molecular systems. Herein we report the first spectroscopic characterization of well-defined paramagnetic bridging hydrides. Time-resolved FTIR spectroelectrochemical experiments on a subsecond time scale revealed that single-electron transfer to the μ-hydride di-iron dithiolate complex 1 generates a 37-electron valence-delocalized species with no gross structural reorganization of the coordination sphere. DFT calculations support and (1)H and (2)H EPR measurements confirmed the formation an S = ½ paramagnetic complex (g = 2.0066) in which the unpaired spin density is essentially symmetrically distributed over the two iron atoms with strong hyperfine coupling to the bridging hydride (A(iso) = -75.8 MHz).

A general approach for the prediction of EPR spectra directly and completely from single dynamical trajectories generated from Molecular Dynamics (MD) simulations is described. The approach is applicable to an arbitrary system of electron and nuclear spins described by a general form of the spin-Hamiltonian for the entire motional range. It is shown that for a reliable simulation of motional EPR spectra only a single truncated dynamical trajectory generated until the point when correlation functions of rotational dynamics are completely relaxed is required. The simulation algorithm is based on a combination of the propagation of the spin density matrix in the Liouville space for this initial time interval and the use of well defined parameters calculated entirely from the dynamical trajectory for prediction of the evolution of the spin density matrix at longer times. A new approach is illustrated with the application to a nitroxide spin label MTSL attached to the protein sperm whale myoglobin. It is shown that simulation of the EPR spectrum, which is in excellent agreement with experiment, can be achieved from a single MD trajectory. Calculations reveal the complex nature of the dynamics of a spin label which is a superposition of the fast librational motions within dihedral states, of slow rotameric dynamics among different conformational states of the nitroxide tether and of the slow rotational diffusion of the protein itself. The significance of the slow rotameric dynamics of the nitroxide tether on the overall shape of the EPR spectrum is analysed and discussed.

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.

We report the prediction of motional EPR spectra of the metalloprotein sperm whale myoglobin spin labelled with nitroxide directly from Molecular Dynamics (MD) simulations at the atomistic scale. We show that an accurate simulation of EPR spectra can be achieved from a single MD trajectory which is in excellent agreement with experiment. Simulations have been carried out using a general method reported previously by us for the simulation of EPR spectra form single dynamical trajectories. Our calculations demonstrate the complex nature of the dynamics of a spin label which is a superposition of the fast librational motions around dihedral states, of slow conformational flips among different rotameric states and of the slow rotational diffusion of the protein itself. The MD-EPR methodology reported does not require any additional stochastic modelling using adjustable parameters and opens, for the first time, the prospect of the simulation of EPR spectra entirely from single MD trajectories. Such a technique not only simplifies the interpretation and analysis of EPR spectra but also opens the possibility, for example, of "computer engineering" of spin-labelled proteins with the desired properties prior to actual EPR experiment.

A nitroxide spin label (SL) has been used to probe the electron spin relaxation times and the magnetic states of the oxygen-binding heme-copper dinuclear site in Escherichia coli cytochrome bo(3), a quinol oxidase (QO), in different oxidation states. The spin lattice relaxation times, T(1), of the SL are enhanced by the paramagnetic metal sites in QO and hence show a strong dependence on the oxidation state of the latter. A new, general form of equations and a computer simulation program have been developed for the calculation of relaxation enhancement by an arbitrary fast relaxing spin system of S ≥ 1/2. This has allowed us to obtain an accurate estimate of the transverse relaxation time, T (2), of the dinuclear coupled pair Fe(III)-Cu(B)(II) in the oxidized form of QO that is too short to measure directly. In the case of the F' state, the relaxation properties of the heme-copper center have been shown to be consistent with a ferryl [Fe(IV)=O] heme and Cu(B)(II) coupled by approximately 1.5-3 cm(-1) to a radical. The magnitude suggests that the coupling arises from a radical form of the covalently linked tyrosine-histidine ligand to Cu(II) with unpaired spin density primarily on the tyrosine component. This work demonstrates that nitroxide SLs are potentially valuable tools to probe both the relaxation and the magnetic properties of multinuclear high-spin paramagnetic active sites in proteins that are otherwise not accessible from direct EPR measurements.

Liquid crystals spin their secrets: Electron paramagnetic resonance (EPR) spectra are predicted directly and completely from fully atomistic molecular dynamics (MD) simulations of 4-cyano-4-n-pentylbiphenyl (5CB) nematic liquid crystals with a doped nitroxide spin probe (depicted in yellow; red curve = simulated and blue curve = measured EPR spectrum)., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Magnetic circular dichroism (MCD) is a powerful probe of the electronic and geometric structures of metal centres in metalloproteins. MCD has provided significant insight into the nature of the axial donors at haem centres and, more recently, sophisticated methods for the analysis of MCD spectra have had a major impact on the study of the electronic structures of the ground states of a range of Cu, non-haem iron and Mo-containing active sites. This detail, together with data from other complimentary spectroscopies, has played a major role in defining the chemistry underpinning the catalysis achieved by these metal centres., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

We report simulation of EPR spectra directly and entirely from trajectories generated from molecular dynamics simulations. Results are reported for a model 3beta-DOXYL-5alpha-cholestane spin probe in a coarse-grained solvent representing a 5CB nematic host. The results are in excellent agreement with the experimental spectra. The calculated order parameters associated with the paramagnetic probe show strong correlation with the order parameter of 5CB mesogens and are in agreement with those reported in the literature. Simulation of EPR spectra entirely from molecular dynamics of real structures provides direct correlation between molecular motions and the features observed in the spectra, allowing unambiguous interpretation of the spectra. This method opens the possibility for "computer engineering" of spin-labeled materials with the desired properties, such as spin-labeled proteins, prior to experiment.

In nitrite-treated cytochrome cd1 nitrite reductase, heme d1 is electron paramagnetic resonance silent but paramagnetic. Analysis of the unusual temperature dependence of the magnetic circular dichroism spectra unambiguously demonstrates that the heme d1 is not in the oxoferryl (FeIV=O) state but is low-spin FeIII weakly coupled to a radical species. This species could be either a protein-bound radical generated by a nitrite ion reacting with a heme group resulting in a one-electron oxidation of an amino acid residue, possibly tyrosine or tryptophan, adjacent to heme d1, or a heme d1 FeIIINO complex.

A simple effective method for calculation of EPR spectra from a single truncated dynamical trajectory of spin probe orientations is reported. It is shown that an accurate simulation can be achieved from the small initial fraction of a dynamical trajectory until the point when the autocorrelation function of re-orientational motion of spin label has relaxed. This substantially reduces the amount of time for spectra simulation compared to previous approaches, which require multiple full length trajectories (normally of several microseconds) to achieve the desired resolution of EPR spectra. Our method is applicable to trajectories generated from both Brownian dynamics and molecular dynamics (MD) calculations. Simulations of EPR spectra from Brownian dynamical trajectories under a variety of motional conditions including bi-modal dynamics with different hopping rates between the modes are compared to those performed by conventional method. Since the relatively short timescales of spin label motions are realistically accessible by modern MD computational methods, our approach, for the first time, opens the prospect of the simulation of EPR spectra entirely from MD trajectories of real proteins structures.

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.

A search for conformational changes at the cytosolic entrance to the proton channels of the heme-copper quinol oxidase (QO), cytochrome bo3, E. coli, has been carried out using site directed nitroxide spin labeling (SDSL) of cysteine residues. These were positioned at R134 and R309, on loops that link helices II and III and VI and VII at the entrances to the D and K proton channels, respectively. The motional characteristics of both labels have been determined using X- and W-band EPR spectroscopy at room temperature in selected redox levels in the reaction sequence of QO with oxygen, namely, the mixed valence carbon monoxide form (COMV), the oxidized (O) and super-oxidized (PM) states. The O to PM step is accompanied by the uptake of protons through the K pathway. We find no evidence for changes in the motional characteristics of either label that are expected to be associated with helical motions at the entrances to the channels. Because kinetic studies of mutants show that the redox gating of protons occurs deep within the D channel close to the heme-copper site, the present study implies that no motion is transmitted to the ends of the helices.

The enzyme nitrous oxide reductase (N(2)OR) has a unique tetranuclear copper centre [Cu(4)S], called Cu(Z), at the catalytic site for the two-electron reduction of N(2)O to N(2). The X- and Q-band EPR spectra have been recorded from two forms of the catalytic site of the enzyme N(2)OR from Paracoccus pantotrophus, namely, a form prepared anaerobically, Cu(Z), that undergoes a one-electron redox cycle and Cu(Z)*, prepared aerobically, which cannot be redox cycled. The spectra of both species are axial with that of Cu(Z) showing a rich hyperfine splitting in the g||-region at X-band. DFT calculations were performed to gain insight into the electronic configuration and ground-state properties of Cu(Z) and to calculate EPR parameters. The results for the oxidation state [Cu(+1)(3)Cu(+2)(1)S](3+) are in good agreement with values obtained from the fitting of experimental spectra, confirming the absolute oxidation state of Cu(Z). The unpaired spin density in this configuration is delocalised over four copper ions, thus, Cu(I) 20.1%, Cu(II) 9.5%, Cu(III) 4.8% and Cu(IV) 9.2%, the mu(4)-sulfide ion and oxygen ligand. The three copper ions carrying the highest spin density plus the sulfide ion lie approximately in the same plane while the fourth copper ion is perpendicular to this plane and carries only 4.8% spin density. It is suggested that the atoms in this plane represent the catalytic core of Cu(Z), allowing electron redistribution within the plane during interaction with the substrate, N(2)O.

Fully oxidized cytochrome bo3 from Escherichia coli has been studied in its oxidized and several ligand-bound forms using electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectroscopies. In each form, the spin-coupled high-spin Fe(III) heme o3 and CuB(II) ion at the active site give rise to similar fast-relaxing broad features in the dual-mode X-band EPR spectra. Simulations of dual-mode spectra are presented which show that this EPR can arise only from a dinuclear site in which the metal ions are weakly coupled by an anisotropic exchange interaction of J 1 cm-1. A variable-temperature and magnetic field (VTVF) MCD study is also presented for the cytochrome bo3 fluoride and azide derivatives. New methods are used to extract the contribution to the MCD of the spin-coupled active site in the presence of strong transitions from low-spin Fe(III) heme b. Analysis of the MCD data, independent of the EPR study, also shows that the spin-coupling within the active site is weak with J approximately 1 cm-1. These conclusions overturn a long-held view that such EPR signals in bovine cytochrome c oxidase arise from an S' = 2 ground state resulting from strong exchange coupling (J > 10(2) cm-1) within the active site.

The potential of NIS spectroscopy to study the iron-sulfur clusters in metalloproteins is illustrated using model compounds. The origin of the intense low energy transfer bands is discussed.

The dodecametallic Cr(III) cluster [Cr(12)O(9)(OH)(3)(O(2)CCMe(3))(15)] has a ground spin state of S = 6 characterized by the spin Hamiltonian parameters g(ZZ)() = 1.965, g(XX)() = g(YY)() = 1.960, D(S=)()(6) = +0.088 cm(-)(1), and E(S=)()(6) = 0 (where D and E are the axial and rhombic zero-field splitting parameters, respectively) as determined by multifrequency EPR spectroscopy and magnetization studies. Micro-SQUID magnetization studies reveal steps due to the fine structure of the ground state, with the spacing between the steps in excellent agreement with the D(S=)()(6) value determined by EPR. Analysis of high-resolution optical data (MCD) allows us to determine the single-ion g values and D value (= -1.035 cm(-)(1)) of the constituent Cr(III) ions directly. A vector coupling analysis demonstrates that the cluster ZFS is almost entirely due to the single-ion component. Thus, the relative orientations of the local and cluster magnetic axes can lead to a cluster ZFS of opposite sign to the single-ion value, even when this is the only significant contribution.

The dodecametallic Cr(III) cluster has an S = 6 ground state with an axial zero-field splitting (ZFS) of DS=6 = +0.088 cm-1. Analysis of high-resolution optical data (MCD) allows us to determine the single-ion ZFS of the constituent Cr(III) ions directly (D = -1.035 cm-1). A vector coupling analysis demonstrates that the cluster ZFS is almost entirely single-ion in origin. Thus, the relative orientations of the local and cluster magnetic axes can lead to cluster ZFS of opposite sign to the single-ion even when this is the only significant contribution.

A magneto-optical study has been undertaken of the mixed-valence single-molecule magnet [Mn(IV)(4)Mn(III)(8)O(12)L(16)] in which the ligands, L, are acetate (Mn(12)Ac) or the long-chain carboxylic acid, C(14)H(29)COOH (Mn(12)C(15)), that confers better solubility in organic solvents. Thin polymer films of these compounds in poly(methyl methacrylate) (PMM) have been cast by solvent evaporation to provide samples suitable for variable-temperature and field magnetic circular dichroism (MCD) studies. The absorption spectra in isotropic light are featureless, whereas the low-temperature MCD spectra contain resolved peaks, both positive and negative. MCD magnetization curves measured at temperatures above 4.2 K have established a ground-state spin of S = 10 and an axial zero-field parameter, D, of -0.61 K, similar to that determined for single crystals of Mn(12)Ac. By studying at a variety of optical wavelengths, the polarization ratios of the optical transitions relative to the unique axis of the zero-field distortion have been determined. The MCD magnetization curves measured at 4.2 K between 0 and 5 T for the case of Mn(12)C(15) in the PMM film can be fitted only on the assumption of nonrandom distribution of molecular z-axes arising from stresses in the polymer film during the process of casting. MCD-detected hysteresis curves measured in both frozen solution and PMM films, below the blocking temperature of approximately 3 K, show a high retention of spin polarization after reduction to zero of a polarizing magnetic field. This generates intense zero-field circular dichroism (CD) with maximum intensity for xy-polarized optical transitions whose sign depends on the direction of the original polarizing field. The optical polarization and the selection rules for MCD select a subset of molecular orientations with respect to the direction of field. Thus, the magnetically induced CD provides a highly sensitive and rapid optical method of reading the spin polarization of molecular magnets.

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

Theoretical expressions for magnetic circular dichroism (MCD) of the porphyrin pi-->pi*, spin-allowed charge transfer (CT) and spin-forbidden d-d or CT transitions in high-spin ferric heme are derived. The transitions can be discriminated by their MCD to absorption ratio and/or temperature dependence of MCD intensity. An analysis of the Soret MCD of fluoride complexes of myoglobin (Mb), hemoglobin (Hb) and horseradish peroxidase (HRP), recorded at temperatures from 290 down to 2 K, is given. It is shown that the Soret MCD of HRPF can be well described by overlapping of the pi-->pi* transition with one spin-forbidden CT transition of an 6A1-->4E type. In the case of MbF and HbF it is necessary to assume the presence in the Soret region of the second spin-forbidden CT transition, most probably of an 6A1-->4A1 type. The parameters of transitions have been extracted from a non-linear least-squares fitting procedure. The best fit values of parameter D of the zero-field splitting of the ground manifold for HbF (6.1 cm(-1)) and MbF (6.4 cm(-1)) agree well with those obtained by other methods. The D value for HRPF (8.3 cm(-1)) is obtained for the first time.

We have carried out analysis of the electronic level scheme of the high-spin ferrous hemoproteins by simultaneous fit of the adjustable parameters of a 4-term theoretical model to low-temperature magnetic circular dichroism (MCD), room temperature absorption spectra and available magnetic susceptibility and or Mössbauer data of myoglobin, horseradish peroxidase and cytochrome P450. The high reliability of the ligand field parameter values obtained for deoxymyoglobin is confirmed by good agreement between the predicted and observed magnetic field dependences of MCD and magnetization not used in the fit procedure. In addition, an energy gap between the ground and first excited singlets, estimated to be 4.2 cm-1, agrees well with the value of approximately 4 cm-1 derived from the far-infrared magnetic resonance. Our computer and explicit theoretical analyses give strong evidence that large distinctions in the shape, intensity and temperature behaviour of the MCD of Mb and HRP from those of cytochrome P450 can be described only if the ground manifold in these proteins is 5E eta and 5B2, respectively. The changes in relative energies of the one-electron 3d-orbitals on substitution of an imidazole of histidine for a sulphur anion of cysteine as a protein-derived heme iron ligand are rationalized by the lower ionization potential of the negatively charged sulphur ligand and the higher pi-orbital overlap of its lone pair orbitals with the iron d pi-orbitals compared to the imidazole ligand.