Magnetic Field Effects on the Behavior of Radicals in Protein and DNA Environments

We have examined the behavior of radical pairs derived by hydrogen abstraction of triplet benzophenone and some of its derivatives from bovine serum albumin, human serum albumin and calf thymus DNA. They have been investigated by means of nanosecond laser flash photolysis techniques. The dynamics of radical pair behavior are shown to be sensitive to external magnetic fields; these effects are interpreted using the established model for the influence of magnetic fields on radical pairs in micellar aggregates, in which intersystem crossing of the radical pair is slowed by the external magnetic field. Our results indicate that proteins and DNA can confine the radicals for a sufficiently long period of time for spin evolution to be affected by external fields. In proteins the radical pair retains its geminate character ( i.e . remains confined) for about 0.5–1 μs. Interestingly, the magnetic field effects observed in proteins and in DNA seem to occur in distinct timescales; for example, for 2,3,4,5,6-pentafluorobenzophenone bound to DNA, the magnetic field alters the radical reactivity only over times ≤50 ns, suggesting poor confinement. The timescale for these effects can be increased by promoting Coulombic attraction between DNA and the radical precursor. Electron transfer interactions play a role in the case of DNA.     

Determination of radical re-encounter probability distributions from magnetic field effects on reaction yields

Measurements are reported of the effects of 0-23 mT applied magnetic fields on the spin-selective recombination of Py*- and DMA*+ radicals formed in the photochemical reaction of pyrene and N,N-dimethylaniline. Singlet <--> triplet interconversion in [Py*- DMA*+] radical pairs is probed by investigating combinations of fully protonated and fully deuterated reaction partners. Qualitatively, the experimental B1/2 values for the four isotopomeric radical pairs agree with predictions based on the Weller equation using known hyperfine coupling constants. The amplitude of the "low field effect" (LFE) correlates well with the ratio of effective hyperfine couplings, aDMA/aPy. An efficient method is introduced for calculating the spin evolution of [Py*- DMA*+] radical pairs containing a total of 18 spin-1/2 and spin-1 magnetic nuclei. Quantitative analysis of the magnetic field effects to obtain the radical re-encounter probability distribution f (t )-a highly ill-posed and underdetermined problem-is achieved by means of Tikhonov and maximum entropy regularization methods. The resulting f (t ) functions are very similar for the four isotopomeric radical pairs and have significant amplitude between 2 and 10 ns after the creation of the geminate radical pair. This interval reflects the time scale of re-encounters that are crucial for generating the magnetic field effect. Computer simulations of generalized radical pairs containing six spin-1/2 nuclei show that Weller's equation holds approximately only when the radical pair recombination rate is comparable to the two effective hyperfine couplings and that a substantial LFE requires, but is not guaranteed by, the condition that the two effective hyperfine couplings differ by more than a factor of 5. In contrast, for very slow recombination, essentially any radical pair should show a significant LFE.     

Theory and simulation of anisotropic pair correlations in ferrofluids in magnetic fields

Anisotropic pair correlations in ferrofluids exposed to magnetic fields are studied using a combination of statistical-mechanical theory and computer simulations. A simple dipolar hard-sphere model of the magnetic colloidal particles is studied in detail. A virial-expansion theory is constructed for the pair distribution function (PDF) which depends not only on the length of the pair separation vector, but also on its orientation with respect to the field. A detailed comparison is made between the theoretical predictions and accurate simulation data, and it is found that the theory works well for realistic values of the dipolar coupling constant (λ = 1), volume fraction (φ ≤ 0.1), and magnetic field strength. The structure factor is computed for wavevectors either parallel or perpendicular to the field. The comparison between theory and simulation is generally very good with realistic ferrofluid parameters. For both the PDF and the structure factor, there are some deviations between theory and simulation at uncommonly high dipolar coupling constants, and with very strong magnetic fields. In particular, the theory is less successful at predicting the behavior of the structure factors at very low wavevectors, and perpendicular Gaussian density fluctuations arising from strongly correlated pairs of magnetic particles. Overall, though, the theory provides reliable predictions for the nature and degree of pair correlations in ferrofluids in magnetic fields, and hence should be of use in the design of functional magnetic materials.     

ESR of triplet states in single crystals of photochromic 2,3,4,4-tetrachloro-1-keto-1,4-dihydronaphthalene (β-TKN)

Triplet ESR spectra of irradiated single crystals of β-TKN have been investigated as a function of their orientation with respect to the magnetic field. From the directions of the principal axes of the dipolar coupling tensor and the values of the zero field parameters D = ?96.2 gauss and E = ?2.7 gauss, it follows that the four triplet sites, generated in the crystals, consist of radical pairs. Each pair radicals is formed from two β-TKN molecules, which are nearest neighbours in the lattice. Measurements at the temperatures 4.2 K and 1.2 K show that the triplet state is the ground state of the radical pair.     

Singlet and triplet products of the geminate recombination of a radical pair with a single magnetic nucleus (I = 1/2)

The double-channel recombination and separation of the photochemically created singlet radical pair is investigated, taking into account the spin conversion in a zero magnetic field and the arbitrary initial distance between the radicals. The quantum yields of the singlet and triplet products and the free radicals production are found analytically, assuming that the recombination of the diffusing radicals occurs at contact. All the yields are related to the singlet and triplet populations of the recombining radical pair, subjected to spin conversion and contact exchange interaction. The general analytical expressions for the quantum yields are specified for the particular limits of the weak and strong exchange. They are greatly simplified in the case of polar solvents, especially at the contact start. A close similarity is obtained with the results of a previously developed incoherent model of spin conversion, provided that the conversion rate is appropriately related to the hyperfine coupling constant.     

Transient free radicals in viscous solvents

The majority of free radicals are highly reactive species which participate in bimolecular reactions with each other. Validation of the theory of molecular diffusion and reactivity in the liquid state requires knowledge of rate constants of radical–radical reactions (recombination, disproportionation) and their viscosity dependencies. An accurate comparison of theory and experiment has become available due to experimentally measured diffusion coefficients of reactive radicals by transient grating technique. Initial distribution of radicals in solution can be not random but pair-wise as in photo- or thermoinitiation of free radical polymerization reactions. Probability of a radical escape of a partner (cage escape) characterizes the initiator efficiency. Despite decades of measurement of cage effect values, cage effect dynamics with free radicals have only been investigated quite recently. The present tutorial review considers the effect of viscosity of Newtonian liquid on two types of recombination—in the solvent bulk and in a cage. Further, since radicals are paramagnetic species, external magnetic field affects probability of their reactions in pairs. These effects are also observed in viscous liquids, and reasons for such observations are explained. The recently discovered low magnetic field effect is also observed on radical pairs in viscous liquids.     

Photochemical primary process of photo-Fries rearrangement reaction of 1-naphthyl acetate as studied by MFE probe

Photo-Fries rearrangement reactions of 1-naphthyl acetate (NA) in n-hexane and in cyclohexane were studied by the magnetic field effect probe (MFE probe) under magnetic fields (B) of 0 to 7 T. Transient absorptions of the 1-naphthoxyl radical, T-T absorption of NA, and a short-lifetime intermediate (τ = 24 ns) were observed by a nanosecond laser flash photolysis technique. In n-hexane, the yield of escaped 1-naphthoxyl radicals dropped dramatically upon application of a 3 mT field, but then the yield increased with increasing B for 3 mT < B≤ 7 T. These observed MFEs can be explained by the hyperfine coupling and the Δg mechanisms through the singlet radical pair. The fact that MFEs were observed for the present photo-Fries rearrangement reaction indicates the presence of a singlet radical pair intermediate with a lifetime as long as several tens of nanoseconds.     

The spin mixing process of a radical pair in low magnetic field observed by transient absorption detected nanosecond pulsed magnetic field effect

The spin mixing process of the radical pair in the sodium dodecyl sulfate (SDS) micelle is studied by using a novel technique nanosecond pulsed magnetic field effect on transient absorption. We have developed the equipment for a nanosecond pulsed magnetic field and observed its effect on the radical pair reaction. A decrease of the free radical yield by a reversely directed pulsed magnetic field that cancels static field is observed, and the dependence on its magnitude, which is called pulsed MARY (magnetic field effect on reaction yield) spectra, is studied. The observed spectra reflect the spin mixing in 50-200 ns and show clear time evolution. Theoretical simulation of pulsed MARY spectra based on a single site modified Liouville equation indicates that the fast spin dephasing processes induced by the modulation of electron-electron spin interaction by molecular reencounter affect to the coherent spin mixing by a hyperfine interaction in a low magnetic field.     

Observation of Magnetic Field Effects on Transient Fluorescence Spectra of Cryptochrome 1 From Homing Pigeons

Cryptochromes are suggested to be involved in the bird magnetoreception based on the radical pair mechanism (RPM), a well established theory of weak magnetic field effects on chemical reactions. Two members of cryptochrome/photolyase family were found to respond to magnetic field, however, no direct responses of bird cryptochrome to magnetic field as weak as the Earth's magnetic field have been obtained so far. In this study, we used transient fluorescence spectroscopy to characterize the weak magnetic field effects of bird cryptochromes. To do this, we cloned the cryptochrome 1 gene (clCRY1) from the retina of homing pigeons (Columba livia), expressed it in insect Sf9 cells and analyzed the transient fluorescence of purified clCRY1 by application of 45–300 μT magnetic fields. The flavin adenine dinucleotide (FAD_ox) and glucose oxidase (GOD) in PBS buffer were set as controls which could be excited by light to generate radicals, but would not be sensitive to magnetic field. We observed that the transient fluorescence spectra of clCRY1 were sensitive to the applied magnetic field at room temperature. Our result provides a new proof of the cryptochrome‐based model of avian magnetoreception in vitro.     

Spin dynamics of the radical pair in a low magnetic field studied by the transient absorption detected magnetic field effect on the reaction yield and switched external magnetic field

The spin dynamics of the radical pair generated from the photocleavage reaction of (2,4,6-trimethylbenzoyl)diphenylphosphine oxide (TMDPO) in micellar solutions was studied by the time-resolved magnetic field effect (MFE) on the transient absorption (TA) and by a novel technique, absorption detected switched external magnetic field (AD-SEMF). Thanks to the large hyperfine coupling constant (A = 38 mT), a characteristic negative MFE on the radical yield was observed at a magnetic field lower than 60 mT whereas a positive effect due to the conventional hyperfine (HFM) and relaxation mechanisms (RM) was observed at higher magnetic field. The negative effect can be assigned to the mechanism "so-called" low field effect (LFE) mechanism and has been analyzed thoroughly using a model calculation incorporating a fast spin dephasing process. The time scale of the spin mixing process of LFE studied by AD-SEMF is shorter than the lifetime of the recombination kinetics of the radical pair. These results indicate that the LFE originates from the coherent spin motion. This can be interfered from the fast spin dephasing caused by electron spin interaction fluctuations.     

Control of photo-electron-transfer induced radical production by micellar cages,heavy-atom substituents and magnetic fields

Intramiceller radical pair formation and recombination kinetics in the electron transfer quenching of the thiomine triplet by aniline and various monohalogenated anilines have been studied by micro-second and nanosecond laser flash spectroscopy in reversed micellar solution of CDBA in benzene. Clear kinetic evidence of the micellar cage effect is provided by a comparative spectro-kinetical study in homogeneous aqueous and reversed micellar solution. In zero magnetic field the radical pairs which originate wrth a triplet spin alignment recombine in the waterpools of the micelles with a rate constant of about 3 × 10⁶s^-1 which is not sensitive to the hyperftue or spin-orbit coupling parameters of the aniline-type radical. Long lived radicals are formed by radical escape from the micelles occurring with a rate constant in the-order of 2 × 10⁶s^-1 and being insensitive to an external magnetic field. Intramicellar radical pair recombination is slowed down by an external magnetic field. A maximum effect (measured at 1 T) of a factor of 3 is observed for non-halogenated anilines. Halogen substituation attenuates this magnetic-field effect depending on the strength of spin-orbit coupling exhibited by the halogen substituent. The magnetic-field effect is interpreted in terms of the radical pair mechanism with special emphasis on the role of spin relaxation. Suppression of the magnetic-field effect by halogen substituents is attributed to the spin-rotational relaxation mechanism which is independent of a magnetic field. A heavy-atom suhstituent effect is also borne out in the primary yield of radical pairs which is decreased in the same way as in homogeneous solution. This is atttributed to the role of a triplet exciplex formed as a precursor of the radical pair, where heavy atom substitueuts cause very efficient rediationless decay to the ground state. A magnetic-field effect typical for the triplet mechanism in the exciplex has been detectable with 4-iodoaniline as quencher.     

Theory of magnetic field modulation of radical recombination reactions. II. Short time behavior

The change of spin multiplicity in a radical pair, due to hyperfine interaction and depending on an external magnetic field, is treated by time-dependent perturbation theory. Analytic expressions, valid at short times, but at arbitrary field strengths, are derived which apply to radicals with any given hyperfine structure. The short time region deserves special interest, since here isotope effects in radical reactions, induced by differences in the nuclear magnetic moments rather than in masses, are shown to be much stronger than at longer times.     

Real-time observation of the spin-state mixing process of a micellized radical pair in weak magnetic fields by nanosecond fast field switching

The singlet-triplet spin-state mixing process of a singlet-born radical pair confined in a sodium dodecyl sulfate (SDS) micelle was studied by observing the nanosecond switched external magnetic field (SEMF) effect on the transient absorption signals. A long-lived singlet radical pair is generated by the photoinduced bond cleavage reaction of tetraphenylhydrazine in an SDS micelle. Application of off-on type SEMF results in the increase of the free radical yield contrary to the decrease produced by an applied static magnetic field. The S-T mixing process in low magnetic field was observed by means of a delay-shift SEMF experiment. Observed incoherent mixing processes are explained in terms of the interplay between coherent hyperfine interaction and fast dephasing processes caused by the fluctuation of electron-spin interactions. Singlet-triplet and triplet-triplet dephasing rate constants are determined independently to be 2 x 10(8) and 0.2 x 10(8) s(-)1, respectively, by a simulation based on a modified single-site Liouville equation. This is the first direct observation of the incoherent spin-state mixing process at magnetic fields comparable to the hyperfine interactions of the radical pair.     

A new model for Overhauser enhanced nuclear magnetic resonance using nitroxide radicals

Nitroxide free radicals are the most commonly used source for dynamic nuclear polarization (DNP) enhanced nuclear magnetic resonance (NMR) experiments and are also exclusively employed as spin labels for electron spin resonance (ESR) spectroscopy of diamagnetic molecules and materials. Nitroxide free radicals have been shown to have strong dipolar coupling to (1)H in water, and thus result in large DNP enhancement of (1)H NMR signal via the well known Overhauser effect. The fundamental parameter in a DNP experiment is the coupling factor, since it ultimately determines the maximum NMR signal enhancements which can be achieved. Despite their widespread use, measurements of the coupling factor of nitroxide free radicals have been inconsistent, and current models have failed to successfully explain our experimental data. We found that the inconsistency in determining the coupling factor arises from not taking into account the characteristics of the ESR transitions, which are split into three (or two) lines due to the hyperfine coupling of the electron to the (14)N nuclei (or (15)N) of the nitric oxide radical. Both intermolecular Heisenberg spin exchange interactions as well as intramolecular nitrogen nuclear spin relaxation mix the three (or two) ESR transitions. However, neither effect has been taken into account in any experimental studies on utilizing or quantifying the Overhauser driven DNP effects. The expected effect of Heisenberg spin exchange on Overhauser enhancements has already been theoretically predicted and observed by Bates and Drozdoski [J. Chem. Phys. 67, 4038 (1977)]. Here, we present a new model for quantifying Overhauser enhancements through nitroxide free radicals that includes both effects on mixing the ESR hyperfine states. This model predicts the maximum saturation factor to be considerably higher by the effect of nitrogen nuclear spin relaxation. Because intramolecular nitrogen spin relaxation is independent of the nitroxide concentration, this effect is still significant at low radical concentrations where electron spin exchange is negligible. This implies that the only correct way to determine the coupling factor of nitroxide free radicals is to measure the maximum enhancement at different concentrations and extrapolate the results to infinite concentration. We verify our model with a series of DNP experimental studies on (1)H NMR signal enhancement of water by means of (14)N as well as (15)N isotope enriched nitroxide radicals.     

Hyperfine coupling dependence of the effects of weak magnetic fields on the recombination reactions of radicals generated from polymerisation photoinitiators

The recombination reactions of free radicals formed from the photolysis of a series of polymerisation photoinitiators were studied using time-resolved infrared spectroscopy. All molecules showed Zeeman magnetic field effects (MFEs) in the field range 0-37 mT and those molecules that produced radical pairs with average hyperfine couplings greater than 5 mT showed substantial inverted field effects at fields of less than 10 mT (so-called low field effects, LFEs). Monte Carlo simulations with full treatment of all the isotropic hyperfine couplings in the spin Hamiltonian reproduced well the observed field effects. The use of the usual analysis based on the calculated B1/2 value for the radical pair was found to be inappropriate in systems with substantial LFEs, but simple correlations between this B1/2 value and the observed field features were established.     

Double-channel contact recombination of radical pairs subjected to spin conversion via the Delta g mechanism

The contact recombination from both singlet and triplet states of a radical pair is studied assuming that the spin conversion is carried out by the fast transversal relaxation and Delta g mechanism. The alternative HFI mechanism is neglected as being much weaker in rather large magnetic fields. The magnetic-field-dependent quantum yields of the singlet and triplet recombination products, as well as of the free radical production, are calculated for any initial spin state and arbitrary separation of radicals in a pair. The magnetic field effect is traced and its diffusional (viscosity) dependence is specified.     

Photochemistry of artificial photosynthetic reaction centers in liquid crystals probed by multifrequency EPR (9.5 and 95 GHz)

Photoinduced charge separation and recombination in a carotenoid-porphyrin-fullerene triad C-P-C(60)(1) have been followed by multifrequency time-resolved electron paramagnetic resonance (TREPR) at intermediate magnetic field and microwave frequency (X-band) and high field and frequency (W-band). The electron-transfer process has been characterized in the different phases of two uniaxial liquid crystals (E-7 and ZLI-1167). The triad undergoes photoinduced electron transfer, with the generation of a long-lived charge-separated state, and charge recombination to the triplet state, localized in the carotene moiety, mimicking different aspects of the photosynthetic electron-transfer process. Both the photoinduced spin-correlated radical pair and the spin-polarized recombination triplet are observed starting from the crystalline up to the isotropic phase of the liquid crystals. The W-band TREPR radical pair spectrum has allowed unambiguous assignment of the spin-correlated radical pair spectrum to the charge-separated state C(.+)-P-C(60)(.-). The magnetic interaction parameters have been evaluated by simulation of the spin-polarized radical pair spectrum and the spin-selective recombination rates have been derived from the time dependence of the spectrum. The weak exchange interaction parameter (J = +0.5 +/- 0.2 G) provides a direct measure of the dominant electronic coupling matrix element V between the C(.+)-P-C(60)(.-) radical pair state and the recombination triplet state (3)C-P-C(60). The kinetic parameters have been analyzed in terms of the effect of the liquid crystal medium on the electron-transfer process. Effects of orientation of the molecular triad in the liquid crystal are evidenced by simulations of the carotenoid triplet state EPR spectra at different orientations of the external magnetic field with respect to the director of the mesophase. The order parameter (S = 0.5 +/- 0.05) has been evaluated.     

Contributions of spin-spin interactions to the magnetic field dependence of the triplet quantum yield in photosynthetic reaction centers

Calculations are presented which show that dipolar coupling in the primary radical ion pair of quinone-depleted photosynthetic reaction centers substanually affects the magnetic field dependence of the triplet quantum yield, as does exchange coupling to the semiquinone-Fe(II) complex, when quinone is present. Inclusion of these interactions resolves significant discrepancies between theory and experiment.     

Proton transfer reactions of photogenerated cyanoaromatic-methylaromatic radical ion pairs2

The photochemical reactions of a number of cyanoaromatic (acceptor) and methylaromatic (donor) molecules have been investigated. These reactions can result in the formation of photosubstitution products or benzyl radical coupling products. A survey of our results and previously published data indicates that exergonic photostimulated electron transfer is a necessary but not sufficient condition for the observation of reaction products. The efficiency of proton transfer from the donor cation radical to the acceptor anion radical is determined by the kinetic acidity and basicity of the radical ion pair. Mechanistic evidence is presented which indicates that proton transfer requires diffusion apart and reencounter of the initially formed radical ion pair. Predominant radical pair combination is observed for anion radicals which yield electron-deficient free radicals upon protonation, whereas predominant cage escape and benzyl radical coupling is observed for anion radicals which yield electron-rich free radicals upon protonation.     

Optical Absorption and Magnetic Field Effect Based Imaging of Transient Radicals

Short‐lived radicals generated in the photoexcitation of flavin adenine dinucleotide (FAD) in aqueous solution at low pH are detected with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope. Radicals can be studied under both flash photolysis and continuous irradiation conditions, providing a means of directly probing potential biological magnetoreception within sub‐cellular structures. Direct spatial imaging of magnetic field effects (MFEs) by magnetic intensity modulation (MIM) imaging is demonstrated along with transfer and inversion of the magnetic field sensitivity of the flavin semiquinone radical concentration to that of the ground state of the flavin under strongly pumped reaction cycling conditions. A low field effect (LFE) on the flavin semiquinone–adenine radical pair is resolved for the first time, with important implications for biological magnetoreception through the radical pair mechanism.