Shining a Spotlight on Methyl Groups: Photochemically Induced Dynamic Nuclear Polarization Spectroscopy of 5-Deazariboflavin and Its Nor Analogs.

5-Deazaflavins are analogs of naturally occurring flavin cofactors. They serve as substitutes for natural flavin cofactors to investigate and modify the reaction pathways of flavoproteins. Demethylated 5-deazaflavins are potential candidates for artificial cofactors, allowing us to fine-tune the reaction kinetics and absorption characteristics of flavoproteins. In this contribution, demethylated 5-deazariboflavin radicals are investigated (1) to assess the influence of the methyl groups on the electronic structure of the 5-deazaflavin radical and (2) to explore their photophysical properties with regard to their potential as artificial cofactors. We determined the proton hyperfine structure of demethylated 5-deazariboflavins using photochemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy, as well as density functional theory (DFT). To provide context, we compare our findings to a study of flavin mononucleotide (FMN) derivatives. We found a significant influence of the methylation pattern on the absorption properties, as well as on the proton hyperfine coupling ratios of the xylene moiety, which appears to be solvent-dependent. This effect is enhanced by the replacement of N5 by C5-H in 5-deazaflavin derivatives compared to their respective flavin counterparts.

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).

Magnetic-Field Dependence of LC-Photo-CIDNP in the Presence of Target Molecules Carrying a Quasi-Isolated Spin Pair.

NMR spectroscopy is well known for its superb resolution, especially at high applied magnetic field. However, the sensitivity of this technique is very low. Liquid-state low-concentration photo-chemically-induced dynamic nuclear polarization (LC-photo-CIDNP) is a promising emerging methodology capable of enhancing NMR sensitivity in solution. LC-photo-CIDNP works well on solvent-exposed Trp and Tyr residues, either in isolation or within proteins. This study explores the magnetic-field dependence of the LC-photo-CIDNP experienced by two tryptophan isotopologs in solution upon in situ LED-mediated optical irradiation. Out of the two uniformly 13C,15N-labeled Trp (Trp-U-13C,15N) and Trp-α-13C-ß,ß,2,4,5,6,7-d7 species employed here, only the latter bears a quasi-isolated 1Hα-13Cα spin pair. Computer simulations of the predicted polarization due to geminate recombination of both species display a roughly bell-shaped field dependence. However, while Trp-U-13C,15N is predicted to show a maximum at ca. 500 MHz (11.7 T) and a fairly weak field dependence, Trp-α-13C-ß,ß,2,4,5,6,7-d7 is expected to display a much sharper field dependence accompanied by a dramatic polarization increase at lower field (ca. 200 MHz, 4.7 T). Experimental LC-photo-CIDNP studies on both Trp isotopologs at 1µM concentration, performed at selected fields, are consistent with the theoretical predictions. In summary, this study highlights the prominent field-dependence of LC-photo-CIDNP enhancements (ε) experienced by Trp isotopologs bearing a quasi-isolated spin pair.

Spin Dynamics of Flavoproteins.

This short review reports the surprising phenomenon of nuclear hyperpolarization occurring in chemical reactions, which is called CIDNP (chemically induced dynamic nuclear polarization) or photo-CIDNP if the chemical reaction is light-driven. The phenomenon occurs in both liquid and solid-state, and electron transfer systems, often carrying flavins as electron acceptors, are involved. Here, we explain the physical and chemical properties of flavins, their occurrence in spin-correlated radical pairs (SCRP) and the possible involvement of flavin-carrying SCRPs in animal magneto-reception at earth's magnetic field.

Fragment Screening and Fast Micromolar Detection on a Benchtop NMR Spectrometer Boosted by Photoinduced Hyperpolarization.

Fragment-based drug design is a well-established strategy for rational drug design, with nuclear magnetic resonance (NMR) on high-field spectrometers as the method of reference for screening and hit validation. However, high-field NMR spectrometers are not only expensive, but require specialized maintenance, dedicated space, and depend on liquid helium cooling which became critical over the recurring global helium shortages. We propose an alternative to high-field NMR screening by applying the recently developed approach of fragment screening by photoinduced hyperpolarized NMR on a cryogen-free 80 MHz benchtop NMR spectrometer yielding signal enhancements of up to three orders in magnitude. It is demonstrated that it is possible to discover new hits and kick-off drug design using a benchtop NMR spectrometer at low micromolar concentrations of both protein and ligand. The approach presented performs at higher speed than state-of-the-art high-field NMR approaches while exhibiting a limit of detection in the nanomolar range. Photoinduced hyperpolarization is known to be inexpensive and simple to be implemented, which aligns greatly with the philosophy of benchtop NMR spectrometers. These findings open the way for the use of benchtop NMR in near-physiological conditions for drug design and further life science applications.

Laser- and cryogenic probe-assisted NMR enables hypersensitive analysis of biomolecules at submicromolar concentration.

Solution-state NMR typically requires 100 µM to 1 mM samples. This limitation prevents applications to mass-limited and aggregation-prone target molecules. Photochemically induced dynamic nuclear polarization was adapted to data collection on low-concentration samples by radiofrequency gating, enabling rapid 1D NMR spectral acquisition on aromatic amino acids and proteins bearing aromatic residues at nanomolar concentration, i.e., a full order of magnitude below other hyperpolarization techniques in liquids. Both backbone H1-C13 and side-chain resonances were enhanced, enabling secondary and tertiary structure analysis of proteins with remarkable spectral editing, via the 13C PREPRINT pulse sequence. Laser-enhanced 2D NMR spectra of 5 µM proteins at 600 MHz display 30-fold better S/N than conventional 2D data collected at 900 MHz. Sensitivity enhancements achieved with this technology, denoted as low-concentration photo-CIDNP (LC-photo-CIDNP), depend only weakly on laser intensity, highlighting the opportunity of safer and more cost-effective hypersensitive NMR applications employing low-power laser sources.

Insights Into the Micelle-Induced ß-Hairpin-to-α-Helix Transition of a LytA-Derived Peptide by Photo-CIDNP Spectroscopy.

A choline-binding module from pneumococcal LytA autolysin, LytA239-252, was reported to have a highly stable nativelike ß-hairpin in aqueous solution, which turns into a stable amphipathic α-helix in the presence of micelles. Here, we aim to obtain insights into this DPC-micelle triggered ß-hairpin-to-α-helix conformational transition using photo-CIDNP NMR experiments. Our results illustrate the dependency between photo-CIDNP phenomena and the light intensity in the sample volume, showing that the use of smaller-diameter (2.5 mm) NMR tubes instead of the conventional 5 mm ones enables more efficient illumination for our laser-diode light setup. Photo-CIDNP experiments reveal different solvent accessibility for the two tyrosine residues, Y249 and Y250, the latter being less accessible to the solvent. The cross-polarization effects of these two tyrosine residues of LytA239-252 allow for deeper insights and evidence their different behavior, showing that the Y250 aromatic side chain is involved in a stronger interaction with DPC micelles than Y249 is. These results can be interpreted in terms of the DPC micelle disrupting the aromatic stacking between W241 and Y250 present in the nativelike ß-hairpin, hence initiating conversion towards the α-helix structure. Our photo-CIDNP methodology represents a powerful tool for observing residue-level information in switch peptides that is difficult to obtain by other spectroscopic techniques.

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 9.4 T and PS II at 4.7 T 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.

15N photo-CIDNP MAS NMR analysis of reaction centers of Chloracidobacterium thermophilum.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by 15N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a' (Zn-BChl a') (Tsukatani et al. in J Biol Chem 287:5720-5732, 2012). Based upon experimental and quantum chemical 15N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a'. Chl a and 81-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.

Low-cost LED-based Photo-CIDNP Enables Biocompatible Hyperpolarization of 19 F for NMR and MRI at 7†T and 4.7†T.

Substrates containing 19 F can serve as background-free reporter molecules for NMR and MRI. However, in vivo applications are still limited due to the lower signal-to-noise ratio (SNR) when compared with 1 H NMR. Although hyperpolarization can increase the SNR, to date, only photo-chemically induced dynamic nuclear polarization (photo-CIDNP) allows for hyperpolarization without harmful metal catalysts. Photo-CIDNP was shown to significantly enhance 19 F NMR signals of 3-fluoro-DL-tyrosine in aqueous solution using flavins as photosensitizers. However, lasers were used for photoexcitation, which is expensive and requires appropriate protection procedures in a medical or lab environment. Herein, we report 19 F MR hyperpolarization at 4.7 T and 7 T with a biocompatible system using a low-cost and easy-to-handle LED-based set-up. First hyperpolarized 19 F MR images could be acquired, because photo-CIDNP enabled repetitive hyperpolarization without adding new substrates.

Stabilization of a flavoprotein for solid-state photo-CIDNP MAS NMR at room temperature by embedding in a glassy sugar matrix.

Hyperpolarization via the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect can be detected in frozen solutions of electron transfer proteins generating a radical-pair upon illumination. The effect has been observed in various natural photosynthetic reaction centers and in light-oxygen-voltage (LOV) sensing domains incorporating a flavin mononucleotide (FMN) as chromophore. In LOV domains, where a highly conserved cysteine is mutated to a flavin to interrupt its natural photochemistry, a radical-pair is generated by electron transfer from a nearby tryptophan to the photoexcited triplet state of FMN. During the photocycle, both the LOV domain and the chromophore are photochemically degraded, e.g., by the formation of singlet oxygen. This limits the time for collection of hyperpolarized nuclear magnetic resonance (NMR) data. We show that embedding of the protein into a trehalose sugar glass matrix stabilizes the protein for 13C solid-state photo-CIDNP NMR experiments which can be conducted at room temperature in a powder sample. Additionally, this preparation allows for incorporation of high amounts of protein further boosting the intensity of the detected signals from FMN and tryptophan at natural abundance. Signal assignment is aided by quantum chemical calculations of absolute shieldings. The underlying mechanism for the surprising absorption-only signal pattern is not yet understood. Comparison to calculated isotropic hyperfine couplings imply that the enhancement is not due to the classical radical-pair mechanism (RPM). Analysis of the anisotropic hyperfine couplings associated with solid-state photo-CIDNP mechanisms also show no simple correlation, suggesting a more complex underlying mechanism.

New Aspects of the Antioxidant Activity of Glycyrrhizin Revealed by the CIDNP Technique.

Electron transfer plays a crucial role in ROS generation in living systems. Molecular oxygen acts as the terminal electron acceptor in the respiratory chains of aerobic organisms. Two main mechanisms of antioxidant defense by exogenous antioxidants are usually considered. The first is the inhibition of ROS generation, and the second is the trapping of free radicals. In the present study, we have elucidated both these mechanisms of antioxidant activity of glycyrrhizin (GL), the main active component of licorice root, using the chemically induced dynamic nuclear polarization (CIDNP) technique. First, it was shown that GL is capable of capturing a solvated electron, thereby preventing its capture by molecular oxygen. Second, we studied the effect of glycyrrhizin on the behavior of free radicals generated by UV irradiation of xenobiotic, NSAID-naproxen in solution. The structure of the glycyrrhizin paramagnetic intermediates formed after the capture of a solvated electron was established from a photo-CIDNP study of the model system-the dianion of 5-sulfosalicylic acid and DFT calculations.

Enhanced nuclear-spin hyperpolarization of amino acids and proteins via reductive radical quenchers.

Low-concentration photochemically induced dynamic nuclear polarization (LC-photo-CIDNP) has recently emerged as an effective tool for the hyperpolarization of aromatic amino acids in solution, either in isolation or within proteins. One factor limiting the maximum achievable signal-to-noise ratio in LC-photo-CIDNP is the progressive degradation of the target molecule and photosensitizer upon long-term optical irradiation. Fortunately, this effect does not cause spectral distortions but leads to a progressively smaller signal buildup upon long-term data-collection (e.g. 500 nM tryptophan on a 600 MHz spectrometer after ca. 200 scans). Given that it is generally desirable to minimize the extent of photodamage, we report that low-µM amounts of the reductive radical quenchers vitamin C (VC, i.e., ascorbic acid) or 2-mercaptoethylamine (MEA) enable LC-photo-CIDNP data to be acquired for significantly longer time than ever possible before. This approach increases the sensitivity of LC-photo-CIDNP by more than 100%, with larger enhancement factors achieved in experiments involving more transients. Our results are consistent with VC and MEA acting primarily by reducing transient free radicals of the NMR molecule of interest, thus attenuating the extent of photodamage. The benefits of this reductive radical-quencher approach are highlighted by the ability to collect long-term high-resolution 2D 1H-13C LC-photo-CIDNP data on a dilute sample of the drkN SH3 protein (5 µM).

Assignment of NMR resonances of protons covalently bound to photochemically active cofactors in photosynthetic reaction centers by 13C-1H photo-CIDNP MAS-J-HMQC experiment.

Modified versions of through-bond heteronuclear correlation (HETCOR) experiments are presented to take advantage of the light-induced hyperpolarization that occurs on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. Such 13C-1H photo-CIDNP MAS-J-HMQC and photo-CIDNP MAS-J-HSQC experiments are applied to acquire the 2D 13C-1H correlation spectra of selectively 13C-labeled photochemically active cofactors in the frozen quinone-blocked photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (R.) sphaeroides wild-type (WT). Resulting spectra contain no correlation peaks arising from the protein backbone, which greatly simplifies the assignment of aliphatic region. Based on the photo-CIDNP MAS-J-HMQC NMR experiment, we obtained assignment of selective 1H NMR resonances of the cofactors involved in the electron transfer process in the RC and compared them with values theoretically predicted by density functional theory (DFT) calculation as well as with the chemical shifts obtained from monomeric cofactors in the solution. We also compared proton chemical shifts obtained by photo-CIDNP MAS-J-HMQC experiment under continuous illumination with the ones obtained in dark by classical cross-polarization (CP) HETCOR. We expect that the proposed approach will become a method of choice for obtaining 1H chemical shift maps of the active cofactors in photosynthetic RCs and will aid the interpretation of heteronuclear spin-torch experiments.

Improved sensitivity of laser-enhanced 1Hα-13Cα-correlation via suppression of Cα-C' scalar-coupling evolution.

Low-concentration photochemically induced dynamic polarization (LC-photo-CIDNP) enables the spectroscopic analysis of biomolecules containing the amino acids Trp and Tyr at sub-micromolar concentration in solution. Typical LC-photo-CIDNP pulse sequences involving 1H-13C correlation, however, perform well in the case of aromatic resonances but display a relatively poor signal-to-noise ratio for 13Cα and 13Cß resonances. Here, we develop a novel pulse sequence denoted as 13C perturbation-recovered selective-pulse photo-CINDP enhanced reverse INEPT, or 13C PRESPRINT, tailored to the LC-photo-CIDNP analysis of 1H-13Cα pairs. Our method, which is based on full suppression of 1-bond Cα-C' scalar-coupling evolution during the constant-time delay, results into a sensitivity improvement by a factor of 2. The enhanced performance of this pulse sequence enabled us to improve the analysis of LC-photo-CIDNP laser-power dependence at very low (200 nM) sample concentration. An improved theoretical model, developed to quantitatively describe this laser-power dependence, shows excellent agreement with our 13C PRESPRINT experimental data.

13C → 1H transfer of light-induced hyperpolarization allows for selective detection of protons in frozen photosynthetic reaction center.

In the present study, we exploit the light-induced hyperpolarization occurring on 13C nuclei due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect to boost the NMR signal intensity of selected protons via inverse cross-polarization. Such hyperpolarization transfer is implemented into 1H-detected two-dimensional 13C-1H correlation magic-angle-spinning (MAS) NMR experiment to study protons in frozen photosynthetic reaction centers (RCs). As a first trial, the performance of such an experiment is tested on selectively 13C labeled RCs from the purple bacteria of Rhodobacter sphaeroides. We observed response from the protons belonging to the photochemically active cofactors in their native protein environment. Such an approach is a potential heteronuclear spin-torch experiment which could be complementary to the classical heteronuclear correlation (HETCOR) experiments for mapping proton chemical shifts of photosynthetic cofactors and to understand the role of the proton pool around the electron donors in the electron transfer process occurring during photosynthesis.

Effect of heavy atoms on photochemically induced dynamic nuclear polarization in liquids.

Given its short hyperpolarization time (∼10-6 s) and mostly non-perturbative nature, photo-chemically induced dynamic nuclear polarization (photo-CIDNP) is a powerful tool for sensitivity enhancement in nuclear magnetic resonance. In this study, we explore the extent of 1H-detected 13C nuclear hyperpolarization that can be gained via photo-CIDNP in the presence of small-molecule additives containing a heavy atom. The underlying rationale for this methodology is the well-known external-heavy-atom (EHA) effect, which leads to significant enhancements in the intersystem-crossing rate of selected photosensitizer dyes from photoexcited singlet to triplet. We exploited the EHA effect upon addition of moderate amounts of halogen-atom-containing cosolutes. The resulting increase in the transient triplet-state population of the photo-CIDNP sensitizer fluorescein resulted in a significant increase in the nuclear hyperpolarization achievable via photo-CIDNP in liquids. We also explored the internal-heavy-atom (IHA) effect, which is mediated by halogen atoms covalently incorporated into the photosensitizer dye. Widely different outcomes were achieved in the case of EHA and IHA, with EHA being largely preferable in terms of net hyperpolarization.

Spin-chemistry concepts for spintronics scientists.

Spin chemistry and spintronics developed independently and with different terminology. Until now, the interaction between the two fields has been very limited. In this review, we compile the two "languages" in an effort to enhance communication. We expect that knowledge of spin chemistry will accelerate progress in spintronics.

Analysis of electron donors in photosystems in oxygenic photosynthesis by photo-CIDNP MAS NMR.

Both photosystem I and photosystem II are considerably similar in molecular architecture but they operate at very different electrochemical potentials. The origin of the different redox properties of these RCs is not yet clear. In recent years, insight was gained into the electronic structure of photosynthetic cofactors through the application of photochemically induced dynamic nuclear polarization (photo-CIDNP) with magic-angle spinning NMR (MAS NMR). Non-Boltzmann populated nuclear spin states of the radical pair lead to strongly enhanced signal intensities that allow one to observe the solid-state photo-CIDNP effect from both photosystem I and II from isolated reaction center of spinach (Spinacia oleracea) and duckweed (Spirodela oligorrhiza) and from the intact cells of the cyanobacterium Synechocystis by (13)C and (15)N MAS NMR. This review provides an overview on the photo-CIDNP MAS NMR studies performed on PSI and PSII that provide important ingredients toward reconstruction of the electronic structures of the donors in PSI and PSII.

Spectral editing through laser-flash excitation in two-dimensional photo-CIDNP MAS NMR experiments.

In solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) MAS NMR experiments, strong signal enhancement is observed from molecules forming a spin-correlated radical pair in a rigid matrix. Two-dimensional (13)C-(13)C dipolar-assisted rotational resonance (DARR) photo-CIDNP MAS NMR experiments have been applied to obtain exact chemical shift assignments from those cofactors. Under continuous illumination, the signals are enhanced via three-spin mixing (TSM) and differential decay (DD) and their intensity corresponds to the electron spin density in pz orbitals. In multiple-(13)C labelled samples, spin diffusion leads to propagation of signal enhancement to all (13)C spins. Under steady-state conditions, direct signal assignment is possible due to the uniform signal intensity. The original intensities, however, are inaccessible and the information of the local electron spin density is lost. Upon laser-flash illumination, the signal is enhanced via the classical radical pair mechanism (RPM). The obtained intensities are related to isotropic hyperfine interactions aiso and both enhanced absorptive and emissive lines can be observed due to differences in the sign of the local isotropic hyperfine interaction. Exploiting the mechanism of the polarization, selectivity can be increased by the novel time-resolved two-dimensional dipolar-assisted rotational resonance (DARR) MAS NMR experiment which simplifies the signal assignment compared to complex spectra of the same RCs obtained by continuous illumination. Here we present two-dimensional time-resolved photo-CIDNP MAS NMR experiments providing both directly: signal assignment and spectral editing by sign and strength of aiso. Hence, this experiment provides a direct key to the electronic structure of the correlated radical pair.