Seesaw ligands enforce a coordination geometry of the two coligands being in a cis configuration in an octahedral environment. While nitrogen-based weak field seesaw ligands are often employed, strong field ligands with mixed nitrogen and phosphine donors are rarely encountered. Herein we report the synthesis of iron(II) dihalide complexes 1 supported by a rigid seesaw PNPN ligand, yielding C2v-symmetric compounds. Halide abstraction resulted in formation of 5-fold coordinated cationic complexes 2. Uptake of π-acidic ligands gave cationic complex 3. Dicationic complex 4, containing two acetonitrile monodentate ligands, was prepared from Fe(OTf)2. The structure of 1 and 3 was studied in solid-state and in solution, including 15N NMR spectroscopy. Furthermore, the application as catalysts in sp2-sp3 Kumada cross-coupling was demonstrated, showing high reactivity with a catalyst loading of 1 mol% in 1 h reaction time at room temperature.

In recent years, a distinct class of prokaryotic DNA photolyases containing a ribolumazine and an iron-sulfur cluster in addition to the catalytically active flavin adenine dinucleotide (FAD) cofactor has been identified: FeS-BCP. Previous studies of the structural, photochemical, enzymatic, and signaling properties have revealed photocatalytic and photoreceptor activities for the FeS-BCP subclade. These findings imply that FeS-BCP functions are coupled to the flavin redox state and modulated by the surrounding micro environment. Here, we employ various spectroscopic techniques to investigate the photochemistry of CryB from Rhodobacter sphaeroides. A combination of time-resolved and steady-state techniques allowed elucidation of the photocycle following light excitation on the nanosecond to minute timescale. An accelerated (<5 ns) deprotonation reaction of the terminal electron donor, tryptophan-338, in comparison to other photolyases and cryptochromes has been found with implications for both biological electron transfer and structure-function relationships while no direct involvement of the aforementioned secondary cofactors could be revealed. The obtained results are substantial for future studies of this distinct subclass and advance our understanding of flavoprotein photochemistry in general.

Given the large hyperfine couplings commonly observed in 19F nuclei within radicals, fluorinated amino acids are promising candidates for investigating the solvent exposure of amino acids and redox-active cofactors in proteins, as well as for identifying photogenerated intramolecular spin-correlated radical pairs in proteins through photochemically induced dynamic nuclear polarization (photo-CIDNP) nuclear magnetic resonance (NMR). By analyzing their photo-CIDNP properties in solution, this work aims to establish the foundation for using fluorinated tryptophan derivatives in photo-CIDNP, including protein studies. Although fluorinated tyrosine derivatives have been used in several photo-CIDNP studies, there are hardly any data for fluorinated tryptophan derivatives. We assume this is due to the significant line broadening of 19F resonances of fluorinated tryptophan derivatives when flavin mononucleotide (FMN) is used as a photosensitizer. We attribute this to a photochemical reaction occurring between FMN and the fluorinated tryptophans. The broadening can be avoided by using fluorescein as a photosensitizer. The hyperfine couplings of 1H and 19F nuclei in the fluorinated tryptophan radicals of the commercially available derivatives 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan were probed by geminate photo-CIDNP. Time-resolved photo-CIDNP was used to investigate the microsecond kinetics of 19F and 1H photo-CIDNP polarization in the 6-fluorotryptophan radical, revealing strongly different time evolutions due to the nuclei's distinct paramagnetic relaxation. Experimental considerations for incorporation into proteins and for photo-CIDNP of such proteins are discussed.

Despite structural similarity to flavins, 5-deazaflavins were long thought to act only as two-electron transfer agents. Recently, photo-chemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy provided conclusive evidence for the 5-deazaflavin mononucleotide (5-deazaFMN) radical under physiological conditions. Here, we comprehensively characterize its hyperfine structure, completing the findings of the previous study. A full characterization of the 5-deazaflavin radical is invaluable in light of the re-evaluation of its biological role and potential applications in catalysis. Using 13C isotopologues of 5-deazaFMN and 7,8-didemethyl-5-deazaFMN, we probe the 13C hyperfine interaction of the 5-deazaFMN radical using photo-CIDNP. Linear fits with predictions from density functional theory calculations reveal similar hyperfine structures for both protonation states. Comparison with the FMN radical highlights the impact of structural modifications on electronic properties.

Optically addressable spin systems have been widely studied for quantum-sensing applications. In this work, we demonstrate that photogenerated spin-correlated radical pairs in certain flavoproteins, cryptochrome and improved light-oxygen-voltage protein, can be manipulated by radio waves. This capability enables magnetic field sensing and spatial modulation of photoluminescence using radiofrequency pulses and magnetic field gradients, establishing proteins as a platform for optically addressable spin systems and radiofrequency-based control of biological processes.

We demonstrate the validity of our approach to deduce, from the anisotropy of quantum oscillations, the geometry of short-lived radical pair intermediates in photosynthesis. A global fit of a two-dimensional W-band (94 GHz) electron paramagnetic resonance (EPR) experiment provides the same global minimum values for the geometry of the A-side radical pair P700•+A1A•- in photosystem I (PSI) as observed in a previous Q-band (34 GHz) EPR study, yet with a significantly increased convergence rate of 62%. This demonstrates that the global fit yields the correct radical pair geometry even at Q-band frequencies. With this information, we revisit our previous Q-band study of the cofactor arrangement of P865•+QA•-, the stabilized charge-separated state in purple bacterial reaction centers (RCs). Analysis of calculated two-dimensional data sets of P865•+QA•- reveals that the quantum oscillation technique is unaffected by a mirror ambiguity in disordered solids and thus can provide unambiguous solutions for all five Euler angles of the radical pair geometry. This enables us to elucidate the QA•- to QB electron transfer step in purple bacterial photosynthesis, the subject of controversial discussions for more than 25 years. Our results show that this electron transfer step involves a gating mechanism requiring a 60° rotation of the headgroup of QA•- in its binding pocket.

Fluorine-containing flavin derivatives can be used as probes in flavin-binding proteins forming radical pairs to exploit the photo-chemically induced dynamic nuclear polarization (photo-CIDNP) effect. Knowledge of the hyperfine structure is crucial for studying the mechanism of intramolecular radical-pair formation in proteins. Transient 19F photo-CIDNP NMR has so far not been used to determine the isotropic hyperfine coupling constants of 19F nuclei. Here, we show that this method provides reliable results by studying three monofluorinated flavin mononucleotide (FMN) derivatives in conjunction with 6-fluoro-tryptophan. Combining this method with transient 1H photo-CIDNP spectroscopy leads to a more accurate interpretation of the intermediate radical species forming a radical pair. The gathered information can be used to identify the most promising FMN derivative for usage as a probe for formation of radical pairs in proteins.

Spin-polarized magnetic systems, generated by the interaction of photoactive molecules with light, play a key role in a wide range of scientific applications. Representative examples are OLEDs, organic photovoltaics, and singlet fission. Further, they are important intermediates in certain biological processes including photosynthesis and, possibly, avian magnetoreception. Transient continuous-wave electron paramagnetic resonance (trEPR) spectroscopy is a powerful tool to reveal the temporal evolution of nonequilibrium spin states, which contains valuable information on any photoinduced dynamic processes occurring in these systems. For the analysis of the recorded trEPR data, simulations are essential. While the simulation of static trEPR spectra is supported well by tools like EasySpin, the simulation of time-resolved trEPR data is less developed. Here, we introduce teacups, a new freely available and well-documented Python-based routine for the simulation of the temporal evolution of trEPR spectra. The internal dynamics of different spin-polarized systems can be analyzed, thereby enhancing our mechanistic understanding. In this manuscript, we explain the theoretical background and provide a description of the features and setup of teacups. Further, a step-by-step example for data analysis is provided.

The magnetic compass sensor in night-migratory songbirds is thought to be a flavin-tryptophan radical pair formed by blue-light excitation of the protein cryptochrome-4a (Cry4a) localized in photoreceptor cells in the birds' retinas. The effects of applied magnetic fields on the photochemistry of purified Cry4a from the migratory European robin are well characterized, but it is less clear what, if anything, distinguishes the magnetic responses of the Cry4a proteins from migratory and nonmigratory species. We present here a detailed study of the magnetic sensitivity of Cry4a from the nonmigratory chicken. The wild-type protein is compared with two mutants in which either Arg317 or Glu320, both close to the tryptophan radical, were replaced by the amino acids Cys and Lys, respectively, found in Cry4a from robins and other night-migratory passerines. These sites had previously been identified as probably facilitating the evolution of an optimized magnetic sensor for nocturnal orientation in songbirds. Neither of these mutations was found to affect the reaction kinetics or magnetic sensitivity of the radical pairs, suggesting that any differences in Cry4a between robin and chicken must stem from their ability to transmit magnetic information, for example via protein-protein interactions. In contrast, a Trp → Phe mutation at the end of the tryptophan-tetrad electron transfer chain in both cryptochromes led to a large increase in magnetic sensitivity, suggesting different sensing and signaling roles for the third and fourth tryptophans.

6,7,8-Trimethyllumazine (TML) is a structural analog of the natural cofactor 6,7-dimethyl-8-ribityllumazine. Under basic conditions, TML undergoes a distinctive disproportionation reaction upon photoexcitation. The transiently formed radical pair can be investigated by photo-chemically induced dynamic nuclear polarization (photo-CIDNP) spectroscopy. In this contribution, the structure of the TML anion is analyzed systematically using NMR spectroscopy. Furthermore, the transiently formed TML radicals are investigated and their hyperfine structures elucidated by 1H and 13C photo-CIDNP spectroscopy. Experimental photo-CIDNP intensities are compared with isotropic hyperfine coupling constants from density functional theory (DFT) calculations. The results confirm the formation of an oxidized TML˙ radical and a reduced TMLH˙- radical, the latter potentially protonated at N1. Comparative analysis reveals a substantially different hyperfine structure of the formed radical species which is rationalized based on calculations of spin density distributions. The results provide important insights into photo-induced one-electron transfer reactions of 6,7-dimethyllumazines and their potential role in redox processes in biological systems. The detection and characterization of the oxidized TML˙ radical is of special interest as this oxidation state has not been satisfactorily described in the literature so far. Thus this contribution advances the understanding of the mechanism of formation and the structure of lumazine radicals.

The mechanism by which cryptochrome (CRY) proteins are capable of sensing weak magnetic fields (e.g., the geomagnetic field: ~50 μT) was suggested to be mediated by spin-correlated radical pairs (SCRPs) comprising a flavin adenine dinucleotide (FAD) radical and a tryptophan (Trp) radical which are formed simultaneously by light-induced electron transfer (ET). Here, we provide evidence for direct photoinduced ET that leads to long-lived SCRPs comprising a flavin (Fl) radical and a guanine (G) radical in flavin-tethered single- and double-stranded DNA oligomers by using time-resolved electron paramagnetic resonance (TREPR) spectroscopy. Transient absorption (TA) spectroscopy and its magnetic field effect (MFE) identified RP generation via a triplet-state precursor, in contrast to RP generation via a singlet-state precursor in CRY. Our findings of RPs in Fl-DNA oligomers having microsecond-long lifetimes and capable of exerting a large MFE at room temperature may significantly impact on our understanding of biological magnetoreception.

Cryptochromes are flavin adenine dinucleotide (FAD)-containing blue-light photoreceptors involved in the regulation of the circadian clock and may play a role in magnetic field sensing. The photochemistry of cryptochromes is based on the isoalloxazine moiety, which can be photoreduced and subsequently reoxidized by an electron acceptor such as oxygen, corresponding to a photo-switch between the dark and signaling state. We replaced the FAD cofactor of Drosophila cryptochrome with a series of FAD cofactors modified at the 7 alpha or 8 alpha positions, in order to modulate the chemical properties of the electron acceptor. These modifications were shown to alter the kinetics of the light-dependent reactions. Notably, 7-halogenated FADs form the signaling state more than six times faster compared to the natural FAD cofactor. The more positive reduction potentials as well as the increased intersystem crossing rates due to heavy halogen atoms were identified as reasons for the altered photochemistry. Both parameters show a linear dependence on the reaction kinetics, according to the Hammett relationship. With this knowledge, the photochemistry of cryptochromes may be modified in a defined way without changing its amino acid sequence.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is a hyperpolarization NMR technique that enhances the resonances of molecules involved in the formation of spin-correlated radical pairs. In this contribution, the method is used to selectively enhance the resonances of solvent-exposed, protein-bound flavins by adding an electron donor (tryptophan) to the sample prior to irradiation. To the best of our knowledge, this method has not been used in this way before. By applying photo-CIDNP to two different flavoproteins with solvent-exposed flavins, namely flavodoxin A from Escherichia coli and lumazine protein (LumP) from Photobacterium leiognathi in complex with riboflavin (riboflavin-LumP), we investigate the requirements for radical pair formation. The results reveal that only riboflavin-LumP shows an observable photo-CIDNP effect. Using continuous-wave photo-CIDNP on 1H and 13C nuclei, flavin resonances can be selectively hyperpolarized. Signal assignment is possible by comparing hyperfine data from time-resolved photo-CIDNP and density functional theory (DFT). In addition, the anionic riboflavin radical is determined as the radical present in the geminate radical pair.

This work comprises the synthesis and characterization of aminophosphine (PN)-derived Mn(I) carbonyl complexes and the preliminary investigation of their alkylated congeners for catalytic applications. The complexes fac-[Mn(PN)(CO)3Br] are obtained from the reaction of Mn(CO)5Br with the bidentate ligand PN = R2N(CH2)2PR'2, where R = Me, Et, and pyrrolidine and R' = Ph, iPr, and Cy. Treatment of fac-[Mn(PN)(CO)3Br] with AgOTf yields fac-[Mn(PN)(CO)3OTf]. Upon reaction of fac-[Mn(PN)(CO)3OTf] with MeLi (R' = alkyl) or MeMgCl (R' = aryl), fac-[Mn(PN)(CO)3Me] is formed. fac-[Mn(PCyNMe)(CO)3Me] and fac-[Mn(PPhNMe)(CO)3Me] are identified as the best catalysts for the dimerization of phenylacetylene and the hydroboration of 4-chlorostyrene, respectively.

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.

Partial ligand substitution at the iron pentacarbonyl radical cation generates novel half-sandwich complexes of the type [Fe(eta(6)-arene)(CO)(2)]& sdot;(+) (arene=1,3,5-tri-tert-butylbenzene, 1,3,5-trimethylbenzene, benzene and fluorobenzene). Of those, the bulkier 1,3,5-tri-tert-butylbenzene (mes*) derivative [Fe(mes*)(CO)(2)]& sdot;(+) was fully characterized by XRD analysis, IR, NMR, cw-EPR, M & ouml;ssbauer spectroscopy and cyclic voltammetry as the [Al(ORF)(4)](-) (R-F=C(CF3)(3)) salt. Chemical electronation, i. e., the single electron reduction, with decamethylferrocene generates neutral [Fe(mes*)(CO)(2)], whereas further deelectronation under CO-pressure leads to a dicationic three-legged [Fe(mes*)(CO)(3)](2+) salt with [Al(ORF)(4)](-) counterion. The full substitution of the carbonyl ligands in [Fe(CO)(5)]& sdot;(+)[Al(ORF)(4)](-) mainly resulted in disproportionation reactions, giving solid Fe(0) and the dicationic bis-arene salts [Fe(eta(6)-arene)(2)](2+)([Al(ORF)(4)](-))(2) (arene=1,3,5-trimethylbenzene, benzene and fluorobenzene). Only by employing the very large fluoride bridged anion [F-{Al(ORF)(3)}(2)](-), it was possible to isolate an open shell bis-arene cation salt [Fe(C6H6)(2)]& sdot;(+)[F-{Al(ORF)(3)}(2)](-). The highly reactive cation was characterized by XRD analysis, cw-EPR, M & ouml;ssbauer spectroscopy and cyclic voltammetry. The disproportionation of [Fe(C6H6)(2)]& sdot;(+) salts to give solid Fe(0) and [Fe(C6H6)(2)](2+) salts was analyzed by a suitable cycle, revealing that the thermodynamic driving force for the disproportionation is a function of the size of the anion used and the polarity of the solvent.

The room temperature reduction of various nitriles using amine boranes (ABs) catalysed by a manganese(i) alkyl complex is described. Based on experimental findings, a plausible mechanistic scenario is presented. This includes the presence of two catalytic cycles, one for productive reduction of nitriles and one for hydrogen evolution.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyses the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one (SDO) to a conjugated (22, 24)-diene. We suggest an α4β4 architecture of the 440 kDa enzyme, with each subunit covalently binding an FMN cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by SDO but cannot be oxidized even with strong oxidising agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2 (S26DH2). In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.