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Oxidoreductases facilitating electron transfer between molecules are pivotal in metabolic pathways. Flavin-based electron bifurcation (FBEB), a recently discovered energy coupling mechanism in oxidoreductases, enables the reversible division of electron pairs into two acceptors, bridging exergonic and otherwise unfeasible endergonic reactions. This chapter explores the four distinct FBEB complex families and highlights a decade of structural insights into FBEB complexes. In this chapter, we discuss the architecture, electron transfer routes, and conformational changes across all FBEB families, revealing the structural foundation that facilitate these remarkable functions., (© 2024. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

Flavin mononucleotide (FMN) is a dye belonging to the flavin family. These dyes produce photosensitized degradation of organic compounds via reaction with the excited states of the dye or with reactive oxygen species photogenerated from the triplet of the dye. This article presents a new polymeric dye (FMN-CS) composed of the photosensitizer FMN covalently bonded to chitosan polysaccharide (CS). FMN-CS obtained has a molecular weight of 230 × 10 3  g mol -1 and a deacetylation degree of 74.8%. The polymeric dye is an environmentally friendly polymer with spectroscopic and physicochemical properties similar to those of FMN and CS, respectively. Moreover, under sunlight, it is capable of generating 1 O 2 with a quantum yield of 0.31. FMN-CS, like CS, is insoluble in basic media. This allows easy recovery of the polymeric dye once the photosensitized process has been carried out and makes FMN-CS a suitable photosensitizer for the degradation of pollutants in contaminated waters. To evaluate whether FMN-CS may be used for pollutant degradation, the photosensitized degradation of two trihydroxybenzenes by FMN-CS was studied., (© 2023. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

Flavins are photoenzymatic cofactors often exploiting the absorption of light to energize photoinduced redox chemistry in a variety of contexts. Both flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are used for this function. The study of these photoenzymes has been facilitated using flavin analogs. Most of these analogs involve modification of the flavin ring, and there is recent evidence that adenine (Ade)-modified FAD can affect enzyme turnover, but so far this has only been shown for enzymes where the adenine and flavin rings are close to each other in a stacked conformation. FAD is also stacked in aqueous solution, and its photodynamics are quite different from unstacked FAD or FMN. Oxidized photoexcited FAD decays rapidly, presumably through PET with Ade as donor and Fl* as acceptor. Definitive identification of the spectral signatures of Ade ∙+ and Fl ∙- radicals is elusive. Here we use the FAD analog Flavin 1,N 6 -Ethenoadenine Dinucleotide (εFAD) to study how different photochemical outcomes depend on the identity of the Ade moiety in stacked FAD and its analog εFAD. We have used UV-Vis transient absorption spectroscopy complemented by TD-DFT calculations to investigate the excited state evolution of the flavins. In FAD*, no radicals were observed, suggesting that FAD* does not undergo PET. εFAD* kinetics showed a broad absorption band that suggests a charge transfer state exists upon photoexcitation with evidence for radical pair formation. Surprisingly, significant triplet flavin was produced from εFAD* We hypothesize that the dipolar (ε)Ade moieties differentially modulate the singlet-triplet energy gap, resulting in different intersystem crossing rates. The additional electron density on the etheno group of εFAD supplies better orbital overlap with the flavin S 1 state, accelerating charge transfer in that molecule., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

BLUF (blue light sensor using flavin) proteins are the blue light receptors that consist of flavin-binding BLUF domains and functional domains. Upon blue light excitation, the hydrogen bond network around the flavin chromophore changes, and the absorption spectrum in the visible region shifts to red. Light signal received in the BLUF domain is intramolecularly or intermolecularly transmitted to the functional region. In this review, the reactions of three BLUF proteins with similar EAL functional groups within the protein (BlrP1, and YcgF), or with a separated target protein (PapB) are described using time-resolved diffusion technique. The diffusion coefficients (D) of the BLUF domains did not significantly change upon photoexcitation, whereas those of the full-length proteins BlrP1 and YcgF and the PapB-PapA system significantly decreased. The changes in D should be due to diffusion-sensitive conformational changes (DSCC) that alter the friction of diffusion. The time constants of the major D changes of BlrP1 and PapB-PapA were similar (~ 20 ms), although the magnitude of the friction change depended on the proteins. Similarities and differences among the reactions of these proteins were clarified from the viewpoint of DSCC., (© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.)

NewPHL is a recently discovered subgroup of ancestral DNA photolyases. Its domain architecture displays pronounced differences from that of canonical photolyases, in particular at the level of the characteristic electron transfer chain, which is limited to merely two tryptophans, instead of the "classical" three or four. Using transient absorption spectroscopy, we show that the dynamics of photoreduction of the oxidized FAD cofactor in the NewPHL begins similarly as that in canonical photolyases, i.e., with a sub-ps primary reduction of the excited FAD cofactor by an adjacent tryptophan, followed by migration of the electron hole towards the second tryptophan in the tens of ps regime. However, the resulting tryptophanyl radical then undergoes an unprecedentedly fast deprotonation in less than 100 ps in the NewPHL. In spite of the stabilization effect of this deprotonation, almost complete charge recombination follows in two phases of ~ 950 ps and ~ 50 ns. Such a rapid recombination of the radical pair implies that the first FAD photoreduction step, i.e., conversion of the fully oxidized to the semi-quinone state, should be rather difficult in vivo. We hence suggest that the flavin chromophore likely switches only between its semi-reduced and fully reduced form in NewPHL under physiological conditions.

The two-electron reduction potential for a set of 393 flavin derivatives is presented in this article. These derivatives are substituted flavin on carbon 6, 7, 8, and 9 by coinage transition metals (Cu, Ag, and Au) and conjugated double bond hydrocarbons; and both groups are examined with and without functional groups such as OH, Cl, CH 3 , COOH, and NO 2 . In order to show the validity of the results, the reduction potential of human life molecules, which have experimental values, such as flavin adenine dinucleotide (FAD) and riboflavin (vitamin B 2 ) is calculated. The experimental value for FAD is - 0.22 V, while the obtained theoretical value is - 0.21 V, and the corresponding values for riboflavin are - 0.18 and - 0.19 V, respectively. Theoretical calculations have been carried out by DFT procedure with a 6-31+G** basis set and BLYP xc-functional for coinage transition metals substitution, and MPW1PW9 xc-functionals for conjugated double bond hydrocarbon substitution. Both xc-functionals are chosen by the DFT calibration procedure.

The bacterium Rhodobacter sphaeroides has a short LOV (light-oxygen-voltage) domain, which is not connected to an effector domain but has an α-helix extension at the N-terminus as well as a helix-turn-helix (HTH) motiv at the C-terminus. These extensions offer possibilities for interactions with effector enzymes or DNA. Whereas many LOV domains show a tendency to form dimers in the light state, RsLOV is unique in that it is a dimer in the dark state but dissociates into monomers after blue-light excitation. We studied the kinetics of this dimerization process by a combination of FRET spectroscopy and stopped-flow experiments with a time resolution of ≈10 ms. Although excitation of the flavin chromophore in dye-labeled LOV domains leads to considerable FRET from flavin to the dye, the typical adduct formation between flavin and a nearby cysteine still occurs with considerable yield. We obtain a rate constant for LOV-LOV dimerization in the range (0.8-1.8) × 105 M-1 s-1, and an equilibrium constant of the dark-state dimer in the range (3.0-7.0) × 10-6 M. Dissociation of the dimers in the light state and reforming of dimers after return to the dark state was monitored using an anti-FRET effect caused by excitonic interaction between dye labels on different monomers. Reforming of the dark state dimers is slower than recovery of the flavin-cysteinyl adduct, indicating that light-induced conformational changes in the LOV domain persist for much longer time than the adduct lifetime.

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.

Previous reports suggest that Alzheimer's disease is protected by cholinesterase inhibitors. We synthesized some isoalloxazine derivatives and evaluated them using in vitro cholinesterase inhibition assay. Two of the compounds (7m and 7q) were figured out as potent cholinesterase inhibitors. They further showed anti-Aβ aggregatory activity in the in vitro assay. The current study deals with the evaluation of neuroprotective potentials of the potent compounds (7m and 7q) using different in vitro and in vivo experiments. The compounds were first assessed for their tendency to cross blood-brain barrier using in vitro permeation assay. They were evaluated using scopolamine-induced amnesic mice model. Additionally, ROS scavenging and anti-apoptotic properties of 7m and 7q were established against Aβ1-42-induced toxicity in rat hippocampal neuronal cells. 7m and 7q were also evaluated using Aβ1-42-induced Alzheimer's rat model. Lastly, their involvement in Wnt/β-catenin pathway was also demonstrated. The results indicated good CNS penetration for 7m and 7q. The neuroprotective effects of 7m and 7q were evidenced by improved cognitive ability in both scopolamine and Aβ1-42-induced Alzheimer's-like condition in rodents. The in vivo results also confirmed their anti-cholinesterase and anti-oxidant potential. Immunoblot results showed that treatment with 7m and 7q decreased Aβ1-42, p-tau, cleaved caspase-3, and cleaved PARP levels in Aβ1-42-induced Alzheimer's rat brain. Additionally, immunoblot results demonstrated that 7m and 7q activated the Wnt/β-catenin pathway as evidenced by increased p-GSK-3, β-catenin, and neuroD1 levels in Aβ1-42-induced Alzheimer's rat brain. These findings have shown that isoalloxazine derivatives (7m and 7q) could be the potential leads for developing effective drugs for the treatment of AD.

The kinetics of photolysis of riboflavin (RF) in water (pH 7.0) and in organic solvents (acetonitrile, methanol, ethanol, 1-propanol, 1-butanol, ethyl acetate) has been studied using a multicomponent spectrometric method for the assay of RF and its major photoproducts, formylmethylflavin and lumichrome. The apparent first-order rate constants (k obs) for the reaction range from 3.19 (ethyl acetate) to 4.61 × 10(-3) min(-1) (water). The values of k obs have been found to be a linear function of solvent dielectric constant implying the participation of a dipolar intermediate along the reaction pathway. The degradation of this intermediate is promoted by the polarity of the medium. This indicates a greater stabilization of the excited-triplet states of RF with an increase in solvent polarity to facilitate its reduction. The rate constants for the reaction show a linear relation with the solvent acceptor number indicating the degree of solute-solvent interaction in different solvents. It would depend on the electron-donating capacity of RF molecule in organic solvents. The values of k obs are inversely proportional to the viscosity of the medium as a result of diffusion-controlled processes.

Highly resistant endospores may cause severe problems in medicine as well as in the food and packaging industries. We found that bacterial endospores can be inactivated quickly with reactive oxygen species (ROS) that were generated by a new generation of flavin photosensitizers. Flavins like the natural compound vitamin B2 are already known to produce ROS but they show a poor antimicrobial photodynamic killing efficacy due to the lack of positive charges. Therefore we synthesized new flavin photosensitizers that have one (FLASH-01a) or eight (FLASH-07a) positive charges and can hence attach to the negatively charged surface of endospores. In this study we used standardized Bacillus atrophaeus endospores (ATCC 9372) as a biological surrogate model for a proof-of-concept study of photodynamic inactivation experiments using FLASH-01a and FLASH-07a. After incubation of spores with different flavin concentrations, the flavin derivatives were excited with blue light at a light dose of 70 J cm(-2). The inactivation of spores was investigated either in suspension or after attachment to polyethylene terephthalate (PET) surfaces. Incubation of spores suspended in Millipore water with 4 mM FLASH-01a for 10 seconds and irradiation with blue light for 10 seconds caused a biologically relevant decrease of spore survival of 3.5 log10 orders. Using FLASH-07a under the same conditions we achieved a decrease of 4.4 log10 orders. Immobilized spores on PET surfaces were efficiently killed with 7.0 log10 orders using 8 mM FLASH-07a. The total treatment time (incubation + irradiation) was as short as 20 seconds. The results of this study show evidence that endospores can be fastly and effectively inactivated with new generations of flavin photosensitizers that may be useful for industrial or medical applications in the future.

Following the increasing reports of human toxicity and plasmodium resistance to artemisinin and its derivatives, falcipain-2 (FP-2) is now emerging as the choice antimalarial drug target. Coincidentally, FP-2 is the in vivo target of naturally occurring, therapeutically safe flavonoids (stenopalustroside, myricetin, and fisetin) and symplostatin (symplostatin 4) compounds known to exhibit potent in vitro and in vivo antiplasmodial actions. Here, the structural bases for their inhibitory actions have been studied using molecular dynamics simulation. Myricetin and fisetin act as proton transfer tunnel breakers by inserting between His174 and Cys42, which are key active site residues of FP-2, stenopalustroside inhibits the polarization of His174 by Asn173; a major preparatory step for Cys42/His174 proton transfer process. The roles of flavonoids are favored by T-shaped pi-pi interactions with His174. Symplostatin 4 inserts its methyl-methoxylpyrrolinone moiety into the active site where its proton acceptor function prepares Cys42 for nucleophilic attack on the Michael α,β-unsaturated bonds on its 4(S)-amino-2(E)-pentenoate moiety. Further analyses of the structures identified a unique bridge formed on FP-2 active site groove by stenopalustroside and symplostatin 4 during interaction with the sub-site I of FP-2, whereas fisetin preferentially interacts with sub-site II and myricetin interacts with sub-site III residues. Ultimately, symplostatin-4, myricetin, and fisetin were better than stenopalustroside at trapping FP-2 in its inactive state as revealed by comparative RSMD plots with X-ray structures of FP-2 co-crystallized with inhibitors. Comparative estimates of free energy of binding using the Molecular Mechanics-Poisson Boltzmann Surface Area (MMPBSA) method suggested that His174 protonation may further enhance stenopalustroside-FP-2 interaction. The unique binding signatures of the ligands within the FP-2 active site groove and its sub-sites may explain the subtle differences in their IC50 values and their mechanism of inhibition.

In the spectrophotometric assay of multicomponent systems involved in drug degradation studies, some minor or unknown degradation products may be present. These products may interfere in the assay and thus invalidate the results due to their absorption in the range of analytical wavelengths. This interference may be eliminated by the application of an appropriate correction procedure to obtain reliable data for kinetic treatment. The present study is based on the application of linear and non-linear irrelevant absorption corrections in the multicomponent spectrophotometric assay of riboflavin and formylmethylflavin during the photolysis and hydrolysis studies. The correction procedures take into account the interference caused by minor or unknown products and have shown considerable improvement in the assay data in terms of the molar balance. The treatment of the corrected data has led to more accurate kinetic results in degradation studies.

In recent years it has become increasingly evident that prokaryotic organisms can sense and react to light stimuli via a variety of photosensory receptors and signal transduction pathways. There are two main superfamilies of non-membrane-bound photoreceptors: the bilin-binding phytochrome-related proteins based on GAF (cGMP-specific phosphodiesterases, cyanobacterial adenylate cyclases, and transcription activator FhlA) domains (bilin-GAF proteins), and the flavin-binding proteins (FL-Blues), photoperceptive thanks to their LOV (Light, Oxygen and Voltage) and BLUF (Blue Light sensing Using Flavins) domains. In this manuscript we present a comprehensive scenario of the existence of bilin-GAF, LOV and BLUF proteins in the prokaryotic world and inspect possible phylogenetic pathways, also defining novel criteria for identifying gene (and protein) sequences based on experimentally assessed photochemical events. As a whole we have inspected almost 2000 proteins recovered in 985 bacteria and 16 archaea. For LOV and BLUF proteins, ten and, respectively, twelve superconserved amino acids have been identified, which were used as criterion for selection. A similarly strict parameter cannot be applied to the more variegate family of bilin-GAF domains. The co-presence of bilin-GAF and FL-Blues occurs in 22% of the analyzed bacteria, with emphasis on the bilin-GAF/LOV co-presence in cyanobacteria and of bilin-GAF/BLUF in the Bacteroidetes/Chlorobi group. For construction of phylogeny/distance-trees we used the neighboring-method to obtain a branching pattern, limited to photosensing domains. We observed that in many cases organisms belonging to the same phylum are neighbors, but clustering mostly occurs according to the type of functional domain associated with the photosensing modules.

We present here the synthesis of several new isoalloxazine cyclophanes containing electroactive anthraquinones linked by aliphatic chains of different lengths. Such structural changes provide different interchromophoric orientations leading to the tuning of the rate of the photoinduced electron transfer process from the anthraquinone unit towards the isoalloxazine singlet excited state. Molecular modelling studies were undertaken in order to determine the minimal energy of the proposed structures using Monte Carlo calculations (Amber, Macromodel v.8.1). The compounds have been fully characterised by NMR spectroscopy and the solid state structures of some of the macrocycles have been elucidated. The photophysical studies have been carried out in order to investigate the influence of π-π stacking on the optical properties of the macrocycles.

We describe a competitive binding assay for the analysis of single-base mutation by using abasic site (AP site)-specific binding fluorescent ligands and masking ligands bound to the AP site in DNA duplexes. 2-Amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND) can strongly bind to not only cytosine (C), but also thymine (T) opposite an AP site in the DNA duplexes (K(11)/10(7) M(-1): C, 15; T, 7.0). By contrast, in the presence of lumichrome (Lch) as a masking ligand, ATMND shows a clear binding selectivity for C over T (K(11)/10(7) M(-1): C, 7.0; T, 0.57), which is ascribed to the effective masking effect of Lch to the ATMND-T interaction in the AP site-containing DNA duplexes. It was shown that the competitive binding of ATMND and Lch to the AP site allowed the highly selective detection of C-related single-base mutation in DNAs amplified by polymerase chain reaction.

The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.

Green Fluorescent Protein (GFP) and other related fluorescent proteins are generally used as genetically encoded, chemically inert labels in vivo. This review focuses on the emerging application of fluorescent proteins as light-inducible intracellular photochemical partners. The first example of a chemically active GFP-like protein was the phototoxic red fluorescent protein KillerRed, which can be used for precise light-induced killing of cells, protein inactivation, and studying reactive oxygen species signaling in different cellular compartments. Moreover, recent studies revealed that various GFPs can act as light-induced electron donors in photochemical reactions with biologically relevant electron acceptors. These findings have important implications for practical uses of fluorescent proteins as well as for our understanding of the evolution and biology of this protein family.

Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACβ), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACβF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACβF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACβF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACβF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACβF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACβF2 plays a main role in suppressing the quantum efficiency.

Internet has become a central source for information, tools, and services facilitating the work for medicinal chemists and drug discoverers worldwide. In this paper we introduce a web-based public tool, ChemGPS-NP(Web) (http://chemgps.bmc.uu.se), for comprehensive chemical space navigation and exploration in terms of global mapping onto a consistent, eight dimensional map over structure derived physico-chemical characteristics. ChemGPS-NP(Web) can assist in compound selection and prioritization; property description and interpretation; cluster analysis and neighbourhood mapping; as well as comparison and characterization of large compound datasets. By using ChemGPS-NP(Web), researchers can analyze and compare chemical libraries in a consistent manner. In this study it is demonstrated how ChemGPS-NP(Web) can assist in interpreting results from two large datasets tested for activity in biological assays for pyruvate kinase and Bcl-2 family related protein interactions, respectively. Furthermore, a more than 30-year-old suggestion of "chemical similarity" between the natural pigments betalains and muscaflavins is tested.

The electron-transfer activities of flavin and pterin coenzymes can be fine-tuned by coordination of metal ions, protonation and hydrogen bonding. Formation of hydrogen bonds with a hydrogen-bond receptor in metal-flavin complexes is made possible depending on the type of coordination bond that can leave the hydrogen-bonding sites. The electron-transfer catalytic functions of flavin and pterin coenzymes are described by showing a number of examples of both thermal and photochemical redox reactions, which proceed by controlling the electron-transfer reactivity of coenzymes with metal ion binding, protonation and hydrogen bonding.

DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn2+ -1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and L: -tryptophan (ZnL3) or L: -phenylalanine (ZnL4) as artificial DNA photolyases. We have found that (ZnL3)red, whose flavin unit was reduced in situ by Na2S2O4, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL3 [(ZnL3)ox] in the presence of an excess amount of Et3N at pH 11. UV-vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL3)ox was photoreduced by Et3N into its reduced form (ZnL3)red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL3 with those of ZnL4 suggested that a tryptophan unit in ZnL3 contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.

Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.

The photolysis of formylmethylflavin (FMF), a major intermediate in the photodegradation sequence of riboflavin, has been carried out in water (pH 7.0) and in several organic solvents. FMF produces lumichrome (LC) in organic solvents and LC and lumiflavin (LF) in aqueous solution. FMF and its photoproducts have been analysed using a specific multicomponent spectrophotometric method. FMF undergoes a bimolecular redox reaction on photolysis. The second-order rate constants for the reaction range from 0.66 (chloroform) to 2.44 M(-1) s(-1) (water) and are a linear function of the solvent dielectric constant. A plot of ln k against 1/epsilon is linear for the reactions in 1-butanol, 1-propanol, ethanol, methanol, acetonitrile and water (epsilon approximately 17-79) and non-linear in chloroform and dichloroethane (epsilon approximately 5-10) suggesting a change in reaction mechanism in the two regions. This may be explained on the basis of the existence of a dipolar intermediate along the reaction pathway. The rate of photolysis is governed by the solvation of the intermediate and is thus influenced by the dielectric constant of the medium. The solvent effect on the rate of photolysis of FMF has been expressed in terms of the solvent acceptor number. A linear relationship has been found between ln k and the solvent acceptor number.

Molecular structure, spectroscopic and photophysical data for the singlet state of 3-benzyl-lumiflavin in different solvents are presented. Theoretical studies concerning singlet-singlet and triplet-triplet excitation energies were carried out using time-dependent density functional theory (TD-DFT) calculations. These predictions are in good agreement with the experimental results, which reflect the solvent interactions. All the observable singlet-singlet transitions have pi-pi* character. The title compound appears to be an efficient sensitizer of the production of singlet oxygen (phi(Delta)= 0.53). The crystal structure of 3-benzyl-lumiflavin is also presented, along with its solid-state photophysical data.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.

We report the UV-Vis absorption, fluorescence and transient absorption spectra of selected methylalloxazines adsorbed on cellulose from a polar solvent. The ground-state properties of these probe molecules in the cellulose matrix are similar to those in polar protic solvents. Fluorescence decay data allowed identification of three emitting species for every molecule studied, excluding 1-methyllumichrome which lacks the capacity to rearrange into an isoalloxazinic form. The short-lived emission component was attributed to the neutral form of the molecule, and the two longer-lived components were assigned to the two distinct deprotonated monoanionic forms resulting from dissociation at the respective N(3) and N(1) nitrogen atoms. The two monoanions coexist due to their very similar pKa, values. Transient absorption experiments detected two species created by the laser pulse in these systems. The short-lived species was identified as the triplet excited state, and the long-lived species as the semireduced radical, formed by hydrogen atom or proton transfer from the glycosidic unit to the alloxazine carbonyl group.

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

Two new techniques offering considerable improvements in the quality of 1H photo-CIDNP spectra of proteins are demonstrated. Both focus on the problem of progressive photo-degradation of the flavin dye used to generate polarization in exposed tryptophan, tyrosine and histidine side-chains. One approach uses rapid addition and removal of protein/flavin solution between light flashes to mix the NMR sample and introduce fresh dye into the laser-irradiated region. The other involves chemical oxidation of photo-reduced flavin by the addition of hydrogen peroxide. In both cases a larger number of scans can be accumulated before the flavin is exhausted than would otherwise be possible. The techniques are demonstrated by 600 MHz CIDNP-NOESY spectroscopy of bovine holo-alpha-lactalbumin, and by real-time CIDNP observation of the refolding of bovine apo-alpha-lactalbumin following rapid dilution from a high concentration of chemical denaturant.

Photoexcitation of flavin analogs generates the lowest triplet state (via intersystem crossing from the fist excited singlet state) in the nanosecond time domain and with high quantum efficiency. The triplet, being a strong oxidant, can abstract a hydrogen atom (or an electron) from a reduced donor in a diffusion-controlled reaction. If the donor is a redox protein, the oxidation process can be used to initiate an electron transfer sequence involving either intramolecular or intermolecular reactions. If the donor is an organic compound such as EDTA, the neutral flavin semiquinone will be produced by H atom abstraction; this is a strong reductant and can subsequently transfer a hydrogen atom (or an electron) to an oxidized redox protein, thereby again initiating a sequence of intramolecular or intermolecular processes. If flavin photoexcitation is accomplished using a pulsed light source, the initiation of these protein electron transfer reactions can be made to occur in the nanosecond to microsecond time domain, and the sequence of events can be followed by time-resolved spectrophotometry to obtain rate constants and thus mechanistic information. The present paper describes this technology, and selected examples of its use in the investigation of redox protein mechanisms are given.

The release mechanism for ferritin iron and the nature of the compound(s) which donate iron to the mitochondria are two important problems of intracellular iron metabolism which still await their solution. We have previously shown that isolated mitochondria reduce exogenously added flavins in a ubiquinol-flavin oxidoreductase reaction at the C-side of the inner membrane and that the resulting dihydroflavins function as reductants in mitochondrial mobilization of iron from ferritin (Ulvik, R. J., and Romslo, I. (1981). Biochim. Biophys. Acta 635, 457-469). In the present study it is shown that the rate at which iron is removed from ferritin depends on the capability of the flavins to penetrate (1) the mitochondrial outer membrane and (2) the intersubunit channels of the ferritin protein shell. Intact mitochondria reduce flavins at rates which decrease in the following order: riboflavin >> FAD > FMN. The ferritin iron mobilization rates decrease in the order of riboflavin > FMN > FAD. The results are further support for the operation of a flavin-dependent mitochondrial ferrireductase, and strengthen the suggested role for ferritin as a donor of iron to the mitochondria.