Solid-state photo-CIDNP Effect Research Articles

Electronic Structures of Radical-Pair-Forming Cofactors in a Heliobacterial Reaction Center.

Photosynthetic reaction centers (RCs) are membrane proteins converting photonic excitations into electric gradients. The heliobacterial RCs (HbRCs) are assumed to be the precursors of all known RCs, making them a compelling subject for investigating structural and functional relationships. A comprehensive picture of the electronic structure of the HbRCs is still missing. In this work, the combination of selective isotope labelling of 13C and 15N nuclei and the utilization of photo-CIDNP MAS NMR (photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance) allows for highly enhanced signals from the radical-pair-forming cofactors. The remarkable magnetic-field dependence of the solid-state photo-CIDNP effect allows for observation of positive signals of the electron donor cofactor at 4.7 T, which is interpreted in terms of a dominant contribution of the differential relaxation (DR) mechanism. Conversely, at 9.4 T, the emissive signals mainly originate from the electron acceptor, due to the strong activation of the three-spin mixing (TSM) mechanism. Consequently, we have utilized two-dimensional homonuclear photo-CIDNP MAS NMR at both 4.7 T and 9.4 T. These findings from experimental investigations are corroborated by calculations based on density functional theory (DFT). This allows us to present a comprehensive investigation of the electronic structure of the cofactors involved in electron transfer (ET).

Nuclear spin-hyperpolarization generated in a flavoprotein under illumination: experimental field-dependence and theoretical level crossing analysis

The solid-state photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect generates non-equilibrium nuclear spin polarization in frozen electron-transfer proteins upon illumination and radical-pair formation. The effect can be observed in various natural photosynthetic reaction center proteins using magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, and in a flavin-binding light-oxygen-voltage (LOV) domain of the blue-light receptor phototropin. In the latter system, a functionally instrumental cysteine has been mutated to interrupt the natural cysteine-involving photochemistry allowing for an electron transfer from a more distant tryptophan to the excited flavin mononucleotide chromophore. We explored the solid-state photo-CIDNP effect and its mechanisms in phototropin-LOV1-C57S from the green alga Chlamydomonas reinhardtii by using field-cycling solution NMR. We observed the 13C and, to our knowledge, for the first time, 15N photo-CIDNP signals from phototropin-LOV1-C57S. Additionally, the 1H photo-CIDNP signals of residual water in the deuterated buffer of the protein were detected. The relative strengths of the photo-CIDNP effect from the three types of nuclei, 1H, 13C and 15N were measured in dependence of the magnetic field, showing their maximum polarizations at different magnetic fields. Theoretical level crossing analysis demonstrates that anisotropic mechanisms play the dominant role at high magnetic fields.

Photochemically induced dynamic nuclear polarization NMR on photosystem II: donor cofactor observed in entire plant

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect allows for increase of signal and sensitivity in magic-angle spinning (MAS) NMR experiments. The effect occurs in photosynthetic reaction centers (RC) proteins upon illumination and induction of cyclic electron transfer. Here we show that the strength of the effect allows for observation of the cofactors forming the spin-correlated radical pair (SCRP) in isolated proteins, in natural photosynthetic membranes as well as in entire plants. To this end, we measured entire selectively 13C isotope enriched duckweed plants (Spirodela oligorrhiza) directly in the MAS rotor. Comparison of 13C photo-CIDNP MAS NMR spectra of photosystem II (PS2) obtained from different levels of RC isolation, from entire plant to isolated RC complex, demonstrates the intactness of the photochemical machinery upon isolation. The SCRP in PS2 is structurally and functionally very similar in duckweed and spinach (Spinacia oleracea). The analysis of the photo-CIDNP MAS NMR spectra reveals a monomeric Chl a donor. There is an experimental evidence for matrix involvement, most likely due to the axial donor histidine, in the formation of the SCRP. Data do not suggest a chemical modification of C-131 carbonyl position of the donor cofactor.

15N photo-CIDNP MAS NMR on both photosystems and magnetic field-dependent 13C photo-CIDNP MAS NMR in photosystem II of the diatom Phaeodactylum tricornutum.

Diatoms contribute about 20-25% to the global marine productivity and are successful autotrophic players in all aquatic ecosystems, which raises the question whether this performance is caused by differences in their photosynthetic apparatus. Photo-CIDNP MAS NMR presents a unique tool to obtain insights into the reaction centres of photosystems (PS), by selective enhancement of NMR signals from both, the electron donor and the primary electron acceptor molecules. Here, we present the first observation of the solid-state photo-CIDNP effect in the pennate diatoms. In comparison to plant PSs, similar spectral patterns have been observed for PS I at 9.4T and PS II at 4.7T in the PSs of Phaeodactylum tricornutum. Studies at different magnetic fields reveal a surprising sign change of the 13C photo-CIDNP MAS NMR signals indicating an alternative arrangement of cofactors which allows to quench the Chl a donor triplet state in contrast to the situation in plant PS II. This unusual quenching mechanism is related to a carotenoid molecule in close vicinity to the Chl a donor. In addition to the photo-CIDNP MAS NMR signals arising from the donor and the primary electron acceptor cofactors, a complete set of signals of the imidazole ring ligating to the magnesium of Chl a can be observed.

Same spectral signature in liquid-state and solid-state 1H photo-CIDNP NMR spectra of cyclohexanone

ABSTRACTFor the first time, a 1H solid-state photo-CIDNP effect is reported. The effect is observed in frozen solution of cyclohexanone in 1,4-dioxane-d8 under magic-angle spinning and continuous irradiation by the full spectrum of a xenon arc lamp. The spectral features show close similarity with those observed by 1H liquid-state photo-CIDNP NMR.

Photo-CIDNP in the Reaction Center of the Diatom Cyclotella meneghiniana Observed by 13C MAS NMR

Abstract Photo-CIDNP MAS NMR presents a unique tool to obtain insight into the photosynthetic reaction centers (RCs) of bacteria and plants. Using the dramatic enhancement of sensitivity and selectivity of the solid-state photo-CIDNP effect, structural as well as functional information can be obtained from the cofactor molecules forming a light-induced spin-correlated radical pair (SCRP) in a given reaction center. Here we demonstrate that the effect can be observed in a further species, which belongs neither to the plant nor the bacteria kingdom. Cyclotella (C.) meneghiniana is a member of the diatom phylum and, therefore, belongs to the kingdom of chromista. Chromista are some of the most productive organisms in nature, even in comparison to trees and terrestrial grasses. The observation of the effect in chromista indicates that the effect occurs in all photosynthetic organisms and completes the list with the last phototrophic kingdoms. Our data also demonstrate that the photo- and spin-chemical machineries of photosystem I of plants and chromista are very similar with respect to structure as well as function.

ChemInform Abstract: Analysis of Electron Donors in Photosystems in Oxygenic Photosynthesis by Photo‐CIDNP MAS NMR

AbstractReview: 80 refs.

The Solid-State Photo-CIDNP Effect

The Solid-State Photo-CIDNP Effect

Photochemically Induced Dynamic Nuclear Polarization Observed by Solid-State NMR in a Uniformly (13)C-Isotope-Labeled Photosynthetic Reaction Center.

A sample of solubilized and quinone-depleted reaction centers from the purple bacterium Rhodobacter (R.) sphaeroides wild type has been prepared entirely (13)C and (15)N isotope labeled at all positions of the protein as well as of the cofactors. In this sample, the occurrence of the solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect has been probed by (13)C solid-state magic-angle spinning NMR under illumination. Under continuous illumination, signal intensities are modified by the three-spin mixing (TSM) mechanism. Time-resolved illumination experiments reveal the occurrence of light-induced nuclear polarization on the time scale of hundreds of microseconds, initially dominated by the transient polarization of the singlet branch of the radical-pair mechanism. A first kinetic analysis shows that the lifetime of the polarization from the singlet branch, indicated by the enhanced absorptive intensities of the signals from aliphatic carbons, is significantly extended. Upon arrival of the polarization from the triplet decay branch, emissive polarization caused by the TSM mechanism is observed. Also, this arrival is significantly delayed. The decay of TSM polarization occurs in two steps, assigned to intra- and intermolecular spin diffusion.

The field-dependence of the solid-state photo-CIDNP effect in two states of heliobacterial reaction centers

The solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect is studied in photosynthetic reaction centers of Heliobacillus mobilis at different magnetic fields by (13)C MAS (magic-angle spinning) NMR spectroscopy. Two active states of heliobacterial reaction centers are probed: an anaerobic preparation of heliochromatophores ("Braunstoff", German for "brown substance") as well as a preparation of cells after exposure to oxygen ("Grünstoff", "green substance"). Braunstoff shows significant increase of enhanced absorptive (positive) signals toward lower magnetic fields, which is interpreted in terms of an enhanced differential relaxation (DR) mechanism. In Grünstoff, the signals remain emissive (negative) at two fields, confirming that the influence of the DR mechanism is comparably low.

Magnetic Field Dependence of the Solid-State Photo-CIDNP Effect Observed in Phototropin LOV1-C57S

Here we present the magnetic field dependence of the solid-state Photo-CIDNP effect observed in phototropin LOV1-C57S using 13 C magic-angle spinning (MAS) NMR spectroscopy. Both dark and light spectra were measured at 4.7 T (i.e., 200 MHz 1 H frequency) using a spinning frequency of 8 kHz. An Avance 200 MHz spectrometer equipped with 4-mm MAS probe (Bruker, Karlsruhe, Germany) was used for the 13 C MAS NMR experiments. The sample was packed into a 4-mm sapphire rotor and inserted into the MAS probe. For a homogeneous sample distribution against the rotor wall, the sample was frozen at a very low spinning frequency of 500 Hz. Freezing was monitored on the proton tuning channel as a shift of ca. 0.1 MHz. The variable temperature unit on the spectrometer was set to 235 K. The spinning frequency was in- creased to 8 kHz after the sample is completely frozen. This frequency and set temperature were used for all 13 C MAS NMR measurements. A simple Hahn-echo pulse sequence with two-pulse phase modulation proton-decoupling was used. Continu- ous illumination was supplied by a 1 kW xenon lamp. The cycle delay was 2 s, and the measurement time was about 12 h. In the spectrum obtained in the dark, standard broad protein responses appear. Under illumination, several strong additional light-induced signals appeared. In contrast to the entirely emissive (negative) peaks in the photo-CIDNP MAS NMR spectra observed at 2.3 T (i.e., 100 MHz 1 H frequency), the first observation of this effect in a nonphotosynthetic system, the light induced 13 C NMR peaks at 4.7 T show mixed absorptive/emissive enhancement pattern. This pattern is reminiscent of the spectra observed by liquid state photo-CIDNP of a LOV2 sample. The observed solid-state photo-CIDNP effect is strongly magnetic field dependent, and this field-dependence is well distinguished for the various nuclei. This large difference in magnetic field dependence reflects the large variety of hyperfine factors found in this comparable small-sized and asymmetric radical pair. Keywords photo-CIDNP; MAS; NMR; phototropin; LOV

Spin Chemistry: Coherent Spin Dynamics Rules Chemical Reactions.

It is a great honor to edit a special issue of Applied Magnetic Resonance, in particular if the area presented is not magnetic resonance spectroscopy itself. The topic of this special issue, spin chemistry [1], can be considered as upspring of magnetic resonance spectroscopy. On the other hand, the field of spin chemistry contributed significantly to the methodological improvement of both NMR and EPR. Indeed, spin chemistry was born with the discovery of the CIDEP (chemically induced dynamic electron polarization) effect by EPR spectroscopy in 1963 [2], followed soon by the observation of CIDNP (chemically induced dynamic nuclear polarization) in chemical dark [3–6] and light [7] reactions. The explanation of these phenomena was presented as so-called ‘radical pair mechanism’ (RPM) in 1969 [8, 9]. That revolutionary reaction scheme allowed for the first time nuclear spin states to decide on the fate of a chemical reaction. Since reaction enthalpies are dramatically larger than spin energies, such kinetic control mechanism was a big surprise, in particular for chemists. Soon magnetic field effects (MFEs) were observed on chemical reaction systems [10, 11] and discussed in particular on photosynthetic systems [12–14] which led to the theory of spin-correlated radical pairs [15, 16]. Upon discovery of the solid-state photo-CIDNP effect [17], the concept of spin-correlated radical pairs was extended to solid-state conditions by introduction of coherent anisotropic interactions [18–23] and to natural low-field conditions by allowing for S–T mixing [24]. Such coherent spin dynamics at natural conditions might explain the persistence of the solid-state photo-CIDNP effect in natural photosynthesis [25]. Another hot field in which spin-chemical research aims for understanding of natural phenomena is dedicated to light-dependent magnetosensing by animals via a RPM [26–28]. In this special issue of Applied Magnetic Resonance, a range of spin-chemical effects on chemical and biological electron transfer systems and complementary experimental methods, i.e. EPR and NMR, is reported. First, spin-chemical effects in chemical systems are presented: Basu and coworkers [29] observe an MFE on photoinduced electron transfer reactions between three aromatic amines and acridone in ethanol at low fields. Joonwichien et al. [30] describe MFEs on the photodegeneration of methylene blue dye. Bagryanskaya et al. [31] study spin-selective excited state relaxation and reactivity of deoxybenzoin (DOB) and benzophenon (BP) molecules by time-resolved EPR spectroscopy. Van der Est and coworkers [32] discuss the time-resolved EPR signature of spin-selective electron transfer in series of metalloporphyrin pyridyl-linked naphthalene diimides. Second, spin-chemical effects on biological systems are described: Alia and coworkers [33] present photo-CIDNP data on photosystem I of plants. Hirayama et al. [34] discuss whether MFEs affect the light-dependent circadian clock in zebrafish. Third, an improved technique in solid-state NMR, a main tool in spin chemistry, is presented. Song et al. [35] show a new technique allowing for improvement of proton resolution in proteins. Hence, the present issue demonstrates that nowadays spin chemistry is a matured and lively field with increasing impact on both, magnetic resonance spectroscopy as well as general chemistry. I express my gratitude to the authors who have contributed to this special issue. I am also grateful to Prof. K. Salikhov and Dr. L. Mosina. Without their efforts, the publication of this special issue would not have been possible.

N Photo-CIDNP MAS NMR To Reveal Functional Heterogeneity in Electron Donor of Different Plant Organisms.

In plants and cyanobacteria, two light-driven electron pumps, photosystems I and II (PSI, PSII), facilitate electron transfer from water to carbon dioxide with quantum efficiency close to unity. While similar in structure and function, the reaction centers of PSI and PSII operate at widely different potentials with PSI being the strongest reducing agent known in living nature. Photochemically induced dynamic nuclear polarization (photo-CIDNP) in magic-angle spinning (MAS) nuclear magnetic resonance (NMR) measurements provides direct excess to the heart of large photosynthetic complexes (A. Diller, Alia, E. Roy, P. Gast, H.J. van Gorkom, J. Zaanen, H.J.M. de Groot, C. Glaubitz, J. Matysik, Photosynth. Res. 84, 303–308, 2005; Alia, E. Roy, P. Gast, H.J. van Gorkom, H.J.M. de Groot, G. Jeschke, J. Matysik, J. Am. Chem. Soc. 126, 12819–12826, 2004). By combining the dramatic signal increase obtained from the solid-state photo-CIDNP effect with 15N isotope labeling of PSI, we were able to map the electron spin density in the active cofactors of PSI and study primary charge separation at atomic level. We compare data obtained from two different PSI proteins, one from spinach (Spinacia oleracea) and other from the aquatic plant duckweed (Spirodella oligorrhiza). Results demonstrate a large flexibility of the PSI in terms of its electronic architecture while their electronic ground states are strictly conserved.

Solid-State Photo-CIDNP Effect Observed in Phototropin LOV1-C57S by 13C Magic-Angle Spinning NMR Spectroscopy

Until now, the solid-state photo-CIDNP effect, discovered in 1994 by Zysmilich and McDermott, has been observed selectively in photosynthetic systems. Here we present the first observation of this effect in a nonphotosynthetic system, the blue-light photoreceptor phototropin LOV1-C57S using (13)C magic-angle spinning (MAS) NMR.

Observation of the solid-state photo-CIDNP effect in entire cells of cyanobacteria Synechocystis

Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) 13C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis.

Nanosecond-Flash 15N Photo-CIDNP MAS NMR on Reaction Centers of Rhodobacter sphaeroides R26

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is observed with time-resolved 15N magic-angle spinning (MAS) nuclear magnetic resonance (NMR) in uniformly 15N-labeled reaction centers (RCs) of the purple bacterium Rhodobacter (Rb.) sphaeroides R26 under illumination with nanosecond laser flashes. The 15N NMR signals enhanced by the solid-state photo-CIDNP effect are related to electron spin densities in the primary radical pair and allow for reconstruction of the electronic structure at atomic scale. New assignments for the photochemically active nitrogens are proposed based on simulations of the intensities. The ratio of electron spin densities between cofactors PL and PM is observed to be about 4:1. The origin of the high asymmetry is discussed.

Electron−Nuclear Spin Dynamics in a Bacterial Photosynthetic Reaction Center

The solid-state photo-CIDNP effect is known to occur in natural photosynthetic reaction centers (RCs) where it can be observed by magic-angle spinning (MAS) NMR as strong modification of signal intensities under illumination compared to experiments performed in the dark. The origin of the effect has been debated. In this paper, we report time-resolved photo-CIDNP MAS NMR data of reaction centers of quinone depleted Rhodobacter sphaeroides. It is demonstrated that the build-up of nuclear polarization on the primary donor and the bacteriopheophytin acceptor depends on the presence and lifetimes of the molecular triplet states of the donor and carotenoid. Analysis of the data proves that up to three electron−nuclear spin-coupling mechanisms and two transient effects are working concomitantly in the spin-chemical machinery of the reaction center.

The solid-state photo-CIDNP effect

The solid-state photo-CIDNP effect is the occurrence of a non-Boltzmann nuclear spin polarization in rigid samples upon illumination. For solid-state NMR, which can detect this enhanced nuclear polarization as a strong modification of signal intensity, the effect allows for new classes of experiments. Currently, the photo- and spin-chemical machinery of various RCs is studied by photo-CIDNP MAS NMR in detail. Until now, the effect has only been observed at high magnetic fields with 13C and 15N MAS NMR and in natural photosynthetic RC preparations in which blocking of the acceptor leads to cyclic electron transfer. In terms of irreversible thermodynamics, the high-order spin structure of the initial radical pair can be considered as a transient order phenomenon emerging under non-equilibrium conditions and as a first manifestation of order in the photosynthetic process. The solid-state photo-CIDNP effect appears to be an intrinsic property of natural RCs. The conditions of its occurrence seem to be conserved in evolution. The effect may be based on the same fundamental principles as the highly optimized electron transfer. Hence, the effect may allow for guiding artificial photosynthesis.

Characterization of the Primary Radical Pair in Reaction Centers of Heliobacillus mobilis by 13C Photo-CIDNP MAS NMR

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by (13)C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the (13)C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 8(1)-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.