Spin-correlated Radical Pair Research Articles

Cavity enhanced detection methods for probing the dynamics of spin correlated radical pairs in solution

Cavity enhanced absorption spectroscopy (CEAS) combined with phase-sensitive detection is employed to study the effects of static magnetic fields on radical recombination reactions. The chemical system comprises the photochemically generated thionine semiquinone radical and a 1,4-diazabicyclo[2.2.2]octane (DABCO) cationic radical in a micellar solution of sodium dodecyl sulphate. Data obtained using the modulated CEAS technique, describing the magnetic field effect (MFE) on reaction yields, are shown to be superior to those obtained using conventional transient absorption (TA) flash photolysis methods typically employed for these measurements. The high sensitivity afforded by modulated CEAS detection is discussed in terms of the new possibilities it offers such as the measurement of magnetic field effects in real biological systems which have hitherto been largely beyond the detection capabilities of existing techniques.

Investigation of the Stationary and Transient A(1) Radical in Trp --> Phe Mutants of Photosystem I.

Photosystem I (PS I) contains two symmetric branches of electron transfer cofactors. In both the A- and B-branches, the phylloquinone in the A1 site is π-stacked with a tryptophan residue and is H-bonded to the backbone nitrogen of a leucine residue. In this work, we use optical and electron paramagnetic resonance (EPR) spectroscopies to investigate cyanobacterial PS I complexes, where these tryptophan residues are changed to phenylalanine. The time-resolved optical data show that backward electron transfer from the terminal electron acceptors to P700·+ is affected in the A- and B-branch mutants, both at ambient and cryogenic temperatures. These results suggest that the quinones in both branches take part in electron transport at all temperatures. The electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pair P700·+A1·− and the photoaccumulated radical anion A1·−, recorded at cryogenic temperature, allowed the identification of characteristic resonances belonging to protons of the methyl group, some of the ring protons and the proton hydrogen-bonded to phylloquinone in the wild type and both mutants. Significant changes in PS I isolated from the A-branch mutant are detected, while PS I isolated from the B-branch mutant shows the spectral characteristics of wild-type PS I. A possible short-lived B-branch radical pair cannot be detected by EPR due to the available time resolution; therefore, only the A-branch quinone is observed under conditions typically employed for EPR and ENDOR spectroscopies.

Time-Resolved EPR Spectra of Spin-Correlated Radical Pairs: Spectral and Kinetic Modulation Resulting from Electron−Nuclear Hyperfine Interactions

This paper expands the established four-state model of spin-correlated radical pairs (SCRPs) to include local nuclear spins which are ubiquitous in real-world systems and essential for the radical pair intersystem crossing (RP-ISC) mechanism. These nuclei are coupled to the unpaired electron spins by hyperfine interaction and split their electron paramagnetic resonance (EPR) lines. Rather than enumerating all possible nuclear states, an algorithm is devised to sort out the net hyperfine offset 2Q, which, along with the electron spin-spin coupling 2J, characterizes the behavior of SCRPs. Using this algorithm, the EPR spectra of SCRPs coupled to arbitrary nuclear spins can be efficiently simulated with only 2J and the EPR spectra of individual radicals as the inputs. Particularly illustrative is the case of a SCRP resulting from photoinduced electron transfer comprised of a spectrally narrow anion radical signal having small hyperfine splittings and a broad cation radical signal having many large hyperfine splittings and a Gaussian width sigma, where the EPR peak of the anion radical exhibits an effective splitting of 2(1/2)J(2)/sigma. For SCRPs having singlet and triplet pathways for charge recombination, their kinetic behavior is obtained concisely by considering the decay rate constants k(S) and k(T) as imaginary energies, while adhering to the existing derivation of the four-state model. These models are employed to interpret the diverse array of spectral and kinetic modulation patterns observed in the experimental EPR spectra of photogenerated SCRPs and to extract the 2J value, which reflects the donor-acceptor electronic coupling. During the first several hundred nanoseconds following photoexcitation, the spectral and time domain characteristics of the measured time-resolved EPR spectra manifest the consequences of the Uncertainty Principle, and the modulation patterns in either domain result from hyperfine splittings between the unpaired electron and the nuclear spins.

Evaluation of nuclear quadrupole interactions as a source of magnetic anisotropy in the radical pair model of the avian magnetic compass

One of the principal proposed biophysical mechanisms put forward to explain the avian magnetic compass sense centres around magnetically sensitive chemistry. Based on a large number of in vitro studies of the effects of applied magnetic fields on the yields and rates of chemical reactions it has been suggested that the anisotropic magnetic interactions in spin-correlated radical pairs could be the source of the directional information that allows migratory birds to use the Earth's magnetic field as a navigational aid. Here numerical quantum mechanical simulations are employed to explore the possibility that the hitherto neglected nuclear quadrupole interaction may provide directional information in a radical pair magnetoreceptor. It is concluded that although nuclear quadrupole interactions could fulfil this function, they are unlikely to influence significantly the reaction yield anisotropy in the flavin-tryptophan radical pair that has been proposed as the in vivo magnetoreceptor.

What you get out of high-time resolution electron paramagnetic resonance: example from photosynthetic bacteria

The primary energy conversion steps of natural photosynthesis proceed via light-induced radical ion pairs as short-lived intermediates. Time-resolved electron paramagnetic resonance (EPR) experiments of photosynthetic reaction centers monitor the key charge separated state between the oxidized primary electron donor and reduced quinone acceptor, e.g., P(+)(865)Q(-)(A) of purple photosynthetic bacteria. The time-resolved EPR spectra of P(+)(865)Q(-)(A) are indicative of a spin-correlated radical pair that is created from the excited singlet state of P(865) in an ultra-fast photochemical reaction. Importantly, the spin-correlated radical pair nature of the charge separated state is a common feature of all photosynthetic reaction centers, which gives rise to several interesting spin phenomena such as quantum oscillations, observed at short delay times after optical excitation. In this review, we describe details of the quantum oscillation phenomenon and present a complete analysis of the data obtained from the charge separated state of purple bacteria, P(+)(865)Q(-)(A). The analysis and simulation of the quantum oscillations yield the three-dimensional structure of P(+)(865)Q(-)(A) in the photosynthetic membrane on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of Q(-)(A) is essentially the same as in the X-ray structure. However, the head group of Q(-)(A) has undergone a 60° rotation in the ring plane relative to its orientation in the crystal structure. The results are discussed within the framework of the previously suggested conformational gating mechanism for electron transfer from Q(-)(A) to the secondary quinone acceptor Q(B).

Small but Statistically Reliable Magnetic Field Effect Observed in the Recombination of a Non-Correlated Pair of Biologically Relevant Radicals Nitric Oxide and Superoxide Anion

Magnetic and spin effects, well studied in photo- and radiation-generated chemical systems involving spin-correlated radical pairs, are often called upon as possible mechanisms underlying magnetic effects (MFE) in highly complex biological systems. Although several biologically relevant systems do exist for which this had been indeed verified, often the radical pair mechanism is invoked solely based on the presence of radicals, such as nitric oxide, in the system. This logic has three serious problems: the complexity of the real biological systems, the lack of correlation in thus reacting radicals, and their difference from “normal” partners of the spin-correlated pairs. To address all these issues we created a model chemical system of nitric oxide and superoxide radical recombining to produce peroxynitrite, and studied MFE in it.The radicals were produced as a pair via decomposition of 3-morpholinosydnonimine (SIN-1) in aqueous phosphate buffer. MFE was monitored by comparing the efficiency of peroxynitrite production in exposed and otherwise identical control samples with additional temperature controls. We used static magnets with induction 0.5T and 4.7T. No statistically significant effects were found in the field 0.5T and in temperature controls. In magnetic field 4.7T magnetic field effect of (1.8±0.5)% was obtained.The effect is small, as expected for a non-correlated pair, but statistically reliable. It is apparently limited by extremely fast relaxation of nitric oxide in liquid due to unquenched orbital momentum in the diatomic molecule with electronically degenerate ground state, and develops in f-pairs via the Δg mechanism. Any MFE due to radical pair involving nitric oxide in biological system would probably require either rather strong magnetic field in the Tesla range, or some internal enhancer of magnetic field.

Spin-dynamics of the spin-correlated radical pair in photosystem I. Pulsed time-resolved EPR at high magnetic field

Spin-dynamics of the spin-correlated radical pair (SCRP) P(700)(+)A(1A)(-) in the photosystem I (PSI) reaction center protein have been investigated with high-frequency (HF), time-resolved EPR spectroscopy. The superior spectral resolution of HF EPR enables spin-dynamics for both the donor and acceptor radicals in the pair to be monitored independently. Decay constants of each spin were measured as a function of temperature and compared to data obtained at X-band EPR. Relaxation times, T(1), and decay rates, k(S), are the same at both X- and D-band magnetic fields. The spin-dynamics within the radical pair were determined from theoretical simulation of experimental time-resolved HF EPR spectra. At low temperatures, T < 60 K, the decay of the SCRP from the singlet state, k(S), is the predominant process, while at high temperatures, T > 130 K, the T(1) relaxation is much faster than k(S). The recombination rate k(S) was observed to decrease as the temperature is increased. These EPR spectral results are in agreement with previously reported optical measurements of P(700)(+)A(1)(-) radical pair recombination.

Radiofrequency polarization effects in zero-field electron paramagnetic resonance

Optically detected zero-field electron paramagnetic resonance spectroscopy is used to show that weak linearly and circularly polarized radiofrequency magnetic fields affect the recombination reactions of spin-correlated radical pairs to different extents; the spectra are shown to be consistent with the radical pair mechanism.

Charge-Transfer and Spin Dynamics in DNA Hairpin Conjugates with Perylenediimide as a Base-Pair Surrogate

A perylenediimide chromophore (P) was incorporated into DNA hairpins as a base-pair surrogate to prevent the self-aggregation of P that is typical when it is used as the hairpin linker. The photoinduced charge-transfer and spin dynamics of these hairpins were studied using femtosecond transient absorption spectroscopy and time-resolved EPR spectroscopy (TREPR). P is a photooxidant that is sufficiently powerful to quantitatively inject holes into adjacent adenine (A) and guanine (G) nucleobases. The charge-transfer dynamics observed following hole injection from P into the A-tract of the DNA hairpins is consistent with formation of a polaron involving an estimated 3-4 A bases. Trapping of the (A 3-4) (+*) polaron by a G base at the opposite end of the A-tract from P is competitive with charge recombination of the polaron and P (-*) only at short P-G distances. In a hairpin having 3 A-T base pairs between P and G ( 4G), the radical ion pair that results from trapping of the hole by G is spin-correlated and displays TREPR spectra at 295 and 85 K that are consistent with its formation from (1*)P by the radical-pair intersystem crossing mechanism. Charge recombination is spin-selective and produces (3*)P, which at 85 K exhibits a spin-polarized TREPR spectrum that is diagnostic of its origin from the spin-correlated radical ion pair. Interestingly, in a hairpin having no G bases ( 0G), TREPR spectra at 85 K revealed a spin-correlated radical pair with a dipolar interaction identical to that of 4G, implying that the A-base in the fourth A-T base pair away from the P chromophore serves as a hole trap. Our data suggest that hole injection and transport in these hairpins is completely dominated by polaron generation and movement to a trap site rather than by superexchange. On the other hand, the barrier for charge injection from G (+*) back onto the A-T base pairs is strongly activated, so charge recombination from G (or even A trap sites at 85 K) most likely proceeds by a superexchange mechanism.

Magnetic-field effect on the photoactivation reaction of Escherichia coli DNA photolyase

One of the two principal hypotheses put forward to explain the primary magnetoreception event underlying the magnetic compass sense of migratory birds is based on a magnetically sensitive chemical reaction. It has been proposed that a spin-correlated radical pair is produced photochemically in a cryptochrome and that the rates and yields of the subsequent chemical reactions depend on the orientation of the protein in the Earth's magnetic field. The suitability of cryptochrome for this purpose has been argued, in part, by analogy with DNA photolyase, although no effects of applied magnetic fields have yet been reported for any member of the cryptochrome/photolyase family. Here, we demonstrate a magnetic-field effect on the photochemical yield of a flavin-tryptophan radical pair in Escherichia coli photolyase. This result provides a proof of principle that photolyases, and most likely by extension also cryptochromes, have the fundamental properties needed to form the basis of a magnetic compass.

Time-resolved EPR investigation on the photoreactions of vitamin K with antioxidant vitamins in micelle systems

The photochemical reactions of vitamin K (VK) with antioxidant vitamins (vitamin E (VE) and vitamin C (VC)) in aqueous hexadecyltrimethylammonium chloride (CTAC), sodium dodecyl sulfate (SDS), and Triton X-100 micelle systems, and in an aerosol OT (AOT) reversed micelle system were investigated by a time-resolved EPR (TR-EPR). The photolysis of VK with VE in the aqueous micelle solutions gave the TR-EPR spectra having strong intensity and net emissive polarization, suggesting that the excited triplet state of VK ( 3VK *) was rapidly quenched by VE coexisting inside the micelle. On the other hand, the photolysis of VK with VC in the aqueous SDS and CTAC micelle systems gave the spectra having weak intensity, showing that the reaction between 3VK * and VC was inefficient in these micelle systems, probably because 3VK * scarcely diffused out from the micelle. The photolysis of VK with VC in the AOT reversed micelle solution gave the spin-correlated radical pair CIDEP spectrum. The result suggests that the long-lived radical pair was generated from the reaction between 3VK * and VC in the water/oil interface region of the AOT micelle, although one of the reactants dissolved in the oil phase and another did in the separated water phase.

Effect of nitroxide radicals on chemically induced dynamic electron polarization of spin-correlated radical pairs in aqueous micellar solutions of sodium dodecyl sulfate

The multispin systems consisting of spin-correlated radical pairs (SCRPs) and stable nitroxide radicals, localized in micelles of sodium dodecyl sulfate (SDS), were studied by ESR and pulse laser photolysis techniques. In all the systems studied, the stable nitroxide radicals exert no effect on the shape of the ESR spectra of the SCRPs (in particular, on the shape of their antiphase structure) and on the decay kinetics of the ESR signal of the SCRPs. In the SDS micelles, the electron spin polarization transfer from the nonequilibrium electron spin states of the molecular triplets (SCRP precursors) is the most efficient mechanism of generation of the electron spin polarization in nitroxide radicals. The experimental data also show that the nitroxide radicals and SCRP radicals are most probably distributed uniformly in the micellar phase.

Stochastic Liouville equation studies of FT-EPR spectra of singlet–triplet mixing photo-chemically generated radical pair system

A stochastic Liouville equation (SLE) was numerically solved to obtain pulsed Fourier-transform (FT) EPR spectra on a radical pair system created in a photo-induced chemical reaction. Numerical calculations were applied to the photo-chemical reaction of deuterated acetone and 2-propanol at low temperatures. In this reaction system, the antiphase structures of the EPR signals, so called spin-correlated radical pair (SCRP) signals of two identical isopropyl ketyl radicals and spin-polarized free isopropyl ketyl radicals were observed by FT-EPR and continuous wave time-resolved (CW TR) EPR techniques. In the present work, FT-EPR spectra of the antiphase structure signals of the radical pair themselves as well as spin-polarized free radical signals were simulated. Additionally, rising behavior of free radical signals polarized by the radical pair mechanism (RPM) was also clarified. Furthermore two-dimensional (2D) FT-EPR nutation spectra were simulated in the both cases with and without the radical pairs by the use of SLE. In these simulations, strong DC components in the nutation frequency dimension, were well reproduced as was obtained in experiments. It was shown that relaxation during the microwave pulse was essential for the appearance of the DC components.

Orientation-Resolving Pulsed Electron Dipolar High-Field EPR Spectroscopy on Disordered Solids: I. Structure of Spin-Correlated Radical Pairs in Bacterial Photosynthetic Reaction Centers

Distance and relative orientation of functional groups within protein domains and their changes during chemical reactions determine the efficiency of biological processes. In this work on disordered solid-state electron-transfer proteins, it is demonstrated that the combination of pulsed high-field EPR spectroscopy at the W band (95 GHz, 3.4 T) with its extensions to PELDOR (pulsed electron-electron double resonance) and RIDME (relaxation-induced dipolar modulation enhancement) offers a powerful tool for obtaining not only information on the electronic structure of the redox partners but also on the three-dimensional structure of radical-pair systems with large interspin distances (up to about 5 nm). Strategies are discussed both in terms of data collection and data analysis to extract unique solutions for the full radical-pair structure with only a minimum of additional independent structural information. By this novel approach, the three-dimensional structure of laser-flash-induced transient radical pairs P(865)(*+)Q(A)(*-) in frozen-solution reaction centers (RCs) from the photosynthetic bacterium Rhodobacter (Rb.) sphaeroides is solved. The measured positions and relative orientations of the weakly coupled ion radicals P(865)(*+) and Q(A)(*-) are compared with those of the precursor cofactors P865 and QA known from X-ray crystallography. A small but significant reorientation of the reduced ubiquinone QA is revealed and interpreted as being due to the photosynthetic electron transfer. In contrast to the large conformational change of Q(B)(*-) upon light illumination of the RCs, the small light-induced reorientation of Q(A)(*-) had escaped previous attempts to detect structural changes of photosynthetic cofactors upon charge separation. Although small, they still may be of functional importance for optimizing the electronic coupling of the redox partners in bacterial photosynthesis both for the charge-separation and charge-recombination processes.

Magnetic field effects and time-resolved EPR studies on the photogenerated biradicals in phenothiazine–C 60 linked systems: Clarification of the mechanism of novel magnetic field effects by dependences of methylene chain length and temperature

Magnetic field effects (MFEs) on photogenerated biradical in phenothiazine (Ph)–C 60 linked compound with four methylene group (Ph(4)C 60 ) have been investigated. Transient absorption spectra showed photoinduced intramolecular electron-transfer reactions from the Ph to triplet excited state of C 60 ( 3 C 60 ∗ ) . With increasing magnetic field, the decay rate constant of the photogenerated biradical decreased steeply at lower magnetic fields (<0.2 T), and then recovered in the 0.2 T < H < 1.0 T region. Temperature dependence on the reverse phenomenon of the MFEs in Ph(4)C 60 was clearly different from those in other linked compounds ( Ph( n)C 60 ( n = 6, 8, 10, 12)). The present MFEs can be explained by the contribution of not only spin–lattice relaxation mechanism but also spin–spin relaxation mechanism related to ∣2 J∣. In time-resolved EPR measurements, the spectra in Ph( n)C 60 ( n = 4–12) are assigned to spin-correlated radical pairs. The methylene chain dependence of the time-resolved EPR spectra also supports the mechanism suggested in MFEs.

Exploring hyperfine interactions in spin-correlated radical pairs from photosynthetic proteins: High-frequency ENDOR and quantum beat oscillations

Electron paramagnetic resonance (EPR) and related spectroscopic tools remain among the most important probes of structure and function of natural photosynthetic systems. Indeed, the challenging questions in the study of photosynthesis have to a great extent dictated the directions taken in the development of EPR and associated spectroscopies. In this overview we demonstrate, with recent examples from our laboratories, the potential of high-frequency and time-resolved EPR spectroscopy to reveal unique information about electron transfer processes and the structure of photosynthetic systems. A common feature of these experiments is that they probe hyperfine interactions of the spincorrelated radical pair. Thus, the analysis of the results requires consideration of three interacting spins: two correlated electron spins with one nuclear spin. The results illustrate the importance of resolving nuclear hyperfine structure for obtaining details of structure-function relationships in photosynthetic electron transfer.

High-field EPR, ENDOR and ELDOR on bacterial photosynthetic reaction centers

We report on recent 95 and 360 GHz high-field electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and pulsed electron-electron double resonance (PELDOR) studies of wild-type and mutant reaction centers (RCs) from the photosynthetic bacteriumRhodobacter sphaeroides. Taking advantage of the excellent spectral and temporal resolution of EPR at 95 and 360 GHz, the electron-transfer (ET) cofactors radical ions and spin-correlated radical pairs were characterized by theirg- and hyperfine-tensor components, their anisotropicT2 relaxation as well as by the dipolar interaction between P865•+QA•− radical pairs. The goal of these studies is to better understand the dominant factors determining the specificity and directionality of transmembrane ET processes in photosynthetic RC proteins. In particular, our multifrequency experiments elucidate the subtle cofactor-protein interactions, which are essential for fine-tuning the ET characteristics, e.g., the unidirectionality of the light-induced ET pathways along the A branch of the RC protein. By our high-field techniques, frozen-solution RCs of novel site-specific single and double mutants ofR. sphaeroides were studied to modulate the ET characteristics, e.g., even to the extent that dominant B branch ET prevails. The presented multifrequency EPR work culminates in first 360 GHz ENDOR results from organic nitroxide radicals as well as in first 95 GHz high-field PELDOR results from orientationally selected spin-polarized radical pairs P865•+QA•−, which allow to determine the full geometrical structure of the pairs even in frozen-solution RCs.

Calculation of transient CIDEP spectra of spin-correlated radical pairs in nanotubes

The chemically induced dynamic electron polarization (CIDEP) formed by spin-correlated radical pairs in nanotubes is studied theoretically. Numerical solutions of the stochastic Liouville equation of the density operator are obtained by finite-difference methods. The nanotube is modelled as a one-dimensional nanoreactor with the relative motion of the radicals assumed to be free diffusion. The effect of the non-averaged dipole–dipole interaction of the electron spins on the CIDEP formation is studied in detail. Furthermore, the influence of the radical mobility and the nanotube orientation with respect to the external magnetic field are elucidated.

Time-resolved ESR study on a long-lived radical-ion pair formed in the photoinduced electron-transfer reaction of xanthone and N, N-diethylaniline in 2-propanol

A long-lived radical-ion pair formed in the photoinduced electron-transfer reaction of xanthone and N, N-diethylaniline in 2-propanol is studied using a time-resolved ESR method. The spectrum shows the spin-correlated radical pair with emissive spin polarization of the triplet mechanism. According to the analysis, it is concluded that the radical-ion pair formed transiently has a cluster-like structure by the formation of a network with the surrounding solvent molecules in the present particular system.

Spin-correlated radical pairs: the differential effect in high-field ENDOR spectra

A complete theoretical treatment of the new effect observed in high-field (HF) time-resolved electron-nuclear double resonance (ENDOR) spectra of the spin-correlated radical pair (SCRP) state is presented here. This effect was recently reported for the transient charge-separated state P865+QA− in purple photosynthetic reaction center proteins. One characteristic feature of this new phenomenon is the presence of specific, derivative-type lines in the ENDOR spectra. These lines can be explained by taking into account both a correlation between the electron spins of the photogenerated radical pairs and weak spin-spin interactions within these pairs. This correlation separates the emissive and absorptive lines and results in a differential effect. This effect is enhanced at HF, thus simplifying the analytical description of spectral structure, symmetry features, and interaction parameter dependence. The theoretical analysis presented here demonstrates that only particular nuclei which are essentially coupled to both correlated electron spins contribute to the derivative spectra. The differential effect in SCRP ENDOR manifests remarkable sensitivity to very small “secondary” hyperfine interactions that are usually unresolved in conventional thermalized ENDOR spectra. Application of this theory to experimental SCRP ENDOR spectra will offer a unique look at the internal structure of supramolecular complexes and provide a novel approach for probing electron transfer pathways in natural and artificial photosynthetic assemblies.