Expanding Reaction Horizons: Evidence of the 5-Deazaflavin Radical Through Photochemically Induced Dynamic Nuclear Polarization

Deazaflavins are important analogues of the naturally occurring flavins: riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The use of 5-deazaflavin as a replacement coenzyme in a number of flavoproteins has proven particularly valuable in unraveling and manipulating their reaction mechanisms. It was frequently reported that one-electron-transfer reactions in flavoproteins are impeded with 5-deazaflavin as the cofactor. Based on these findings, it was concluded that the 5-deazaflavin radical is significantly less stable compared to the respective flavin semiquinone and quickly re-oxidizes or undergoes disproportionation. The long-standing paradigm of 5-deazaflavin being solely a two-electron/hydride acceptor/donor—“a nicotinamide in flavin clothing”—needs to be re-evaluated now with the indirect observation of a one-electron-reduced (paramagnetic) species using photochemically induced dynamic nuclear polarization (photo-CIDNP) ¹H nuclear magnetic resonance (NMR) under biologically relevant conditions.     

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting Pt^IV and Ru^II complexes into potentially toxic Pt^II or Ru^II?OH₂ species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate Pt^IV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes Pt^IV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of Pt^IV chemotherapeutic agents.     

PHOTOINACTIVATION OF SPINACH NITRATE REDUCTASE SENSITIZED BY FLAVIN MONONUCLEOTIDE. EVIDENCE FOR THE INVOLVEMENT OF SINGLET OXYGEN

Abstract All the activities of the nitrate reductase complex from spinach are irreversibly inactivated by irradiation of the enzyme with blue light in the presence of flavin mononucleotide. The photoinactivation requires oxygen and is prevented by ethylenediaminetetraacetic acid and by reduced nicotinamide adenine dinucleotide, but not by superoxide dismutase plus catalase. On the other hand, the inactivation is markedly enhanced in 77% deuterated water and it is suppressed by the singlet oxygen quenchers azide, histidine and tryptophan. All these results suggest that singlet oxygen generated by light absorption by flavin mononucleotide, rather than excited flavin mononucleotide or other oxygen species, is the primary agent involved in the photooxidative inactivation of the enzyme.     

Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.     

Infrared spectrum and absorption coefficient of the cofactor flavin in water

Flavins play a key role as redox cofactors of enzymes involved in important metabolic processes. Moreover, they undergo photochemical reactions as chromophores in sensors of blue light or magnetic field in many organisms. The reaction mechanisms of flavoproteins have been investigated by infrared spectroscopy and theoretical studies. However, basic information on flavins in the infrared spectral range has been missing, such as absorption spectra in water and absorption coefficients. Here, the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) were investigated in aqueous medium by Fourier transform infrared spectroscopy. Transmission and attenuated total reflection (ATR) configuration were employed in direct comparison. Absorption spectra in the range of 920–1800 cm⁻¹ were determined after accurate subtraction of the contributions from the water vibrations. The important carbonyl vibrations were resolved at 1661 and 1712 cm⁻¹. The absorption spectra may serve as a reference for theoretical and experimental studies on the effect of the microenvironment on the flavin cofactor. Furthermore, the molar absorption coefficient of FAD at 1547 cm⁻¹ was determined to 2200 L mol⁻¹ cm⁻¹ with an integral absorption coefficient of ∼50,000 L mol⁻¹ cm⁻². These values are prerequisite for the determination of reaction yields in flavoproteins from reaction-induced difference spectra.     

Calculating chemically accurate redox potentials for engineered flavoproteins from classical molecular dynamics free energy simulations

The tricyclic isoalloxazine nucleus of the redox cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) acts as an electron sink in life-sustaining biological electron transfer (eT). The functional diversity of flavin-containing proteins (flavoproteins) transcends that of free flavins. A large body of experimental evidence attributes natural control of flavoprotein-mediated eT to tuning of the thermodynamic driving force by the protein environment. Understanding and engineering such modulation by the protein environment of the flavin redox potential (DeltaE(o)) is valuable in biotechnology and device design. In this study we employed classical molecular dynamics free energy simulations (MDFES), within a thermodynamic integration (TI) formalism, to calculate the change in FMN first reduction potential (DeltaDeltaE(o)(ox/sq)) imparted by 6 flavoprotein active site mutations. The combined performance of the AMBER ff03 (protein) and GAFF (cofactor) force fields was benchmarked against experimental data for mutations close to the isoalloxazine re- and si-faces that perturb the wild-type DeltaE(o)(ox/sq) value in Anabaena flavodoxin. The classical alchemical approach used in this study overestimates the magnitude of DeltaE(o) values, in common with other studies. Nevertheless, chemically accurate DeltaDeltaE(o) values--calculated to within 1 kcal mol(-1) of the experimental value--were obtained for five of the six mutations studied. We have shown that this approach is practical for quantitative in silico screening of the effect of mutations on the first reduction potential where experimental values and structural data are available for the wild-type flavoprotein. This approach promises to be useful as an integral part of future interdisciplinary strategies to engineer desired thermodynamic properties in flavoproteins of biotechnological interest.     

A Molecular Mechanics Model for Flavins

Flavin containing molecules form a group of important cofactors that assist a wide range of enzymatic reactions. Flavins use the redox-active isoalloxazine system, which is capable of one- and two-electron transfer reactions and can exist in several protonation states. In this work, molecular mechanics force field parameters compatible with the CHARMM36 all-atom additive force field were derived for biologically important flavins, including riboflavin, flavin mononucleotide, and flavin adenine dinucleotide. The model was developed for important protonation and redox states of the isoalloxazine group. The partial charges were derived using the CHARMM force field parametrization strategy, where quantum mechanics water–solute interactions are used to target optimization. In addition to monohydrate energies and geometries, electrostatic potential around the compound was used to provide additional restraints during the charge optimization. Taking into account the importance of flavin-containing molecules special attention was given to the quality of bonded terms. All bonded terms, including stiff terms and torsion angle parameters, were parametrized using exhaustive potential energy surface scans. In particular, the model reproduces well the butterfly motion of isoalloxazine in the oxidized and reduced forms as predicted by quantum mechanics in gas phase. The model quality is illustrated by simulations of four flavoproteins. Overall, the presented molecular mechanics model will be of utility to model flavin cofactors in different redox states. © 2019 Wiley Periodicals, Inc.     

Short range photoinduced electron transfer in proteins: QM-MM simulations of tryptophan and flavin fluorescence quenching in proteins

Hybrid quantum mechanical-molecular mechanics (dynamics) were performed on flavin reductase (Fre) and flavodoxin reductase (Fdr), both from Escherichia coli. Each was complexed with riboflavin (Rbf) or flavin mononucleotide (FMN). During 50 ps trajectories, the relative energies of the fluorescing state (S₁) of the isoalloxazine ring and the lowest charge transfer state (CT) were assessed to aid prediction of fluorescence lifetimes that are shortened due to quenching by electron transfer from tyrosine. The simulations for the four cases display a wide range in CT–S₁ energy gap caused by the presence of phosphate, other charged and polar residues, water, and by intermolecular separation between donor and acceptor. This suggests that the Gibbs energy change (ΔG₀) and reorganization energy (λ) for the electron transfer may differ in different flavoproteins.     

One- and Two-Electron Reductions in MiniSOG and their Implication in Catalysis**

The unconventional bioorthogonal catalytic activation of anticancer metal complexes by flavin and flavoproteins photocatalysis has been reported recently. The reactivity is based on a two-electron redox reaction of the photoactivated flavin. Furthermore, when it comes to flavoproteins, we recently reported that site mutagenesis can modulate and improve this catalytic activity in the mini Singlet Oxygen Generator protein (SOG). In this paper, we analyze the reductive half-reaction in different miniSOG environments by means of density functional theory. We report that the redox properties of flavin and the resulting reactivity of miniSOG is modulated by specific mutations, which is in line with the experimental results in the literature. This modulation can be attributed to the fundamental physicochemical properties of the system, specifically (i) the competition of single and double reduction of the flavin and (ii) the probability of electron transfer from the protein to the flavin. These factors are ultimately linked to the stability of flavin‘s electron-accepting orbitals in different coordination modes.     

INTERACTION OF DIHYDROOROTATE DEHYDROGENASE WITH LIGHT*

Abstract— Several flavoproteins are known to undergo photoactivated reduction by EDTA. However, the effects of visible light on the non-heme iron containing flavoproteins have not been characterized previously. Dihydroorotate dehydrogenase was studied as an example of this class of enzymes. Interaction with visible light was found to be complex. Under low intensity photoirradiation of long duration the anaerobic enzyme was partially reduced to a form having increased absorbance at 630 nm. Similar absorbance changes have been correlated with semiquinone species. However, the irradiated enzyme exhibited irreversible changes in catalytic function. Activity with NADH was greatly reduced and a portion of the flavin coenzyme content was labilized. Fluorescence intensity of the enzyme was markedly increased by exposure to light, confirming partial degradation of a catalytic site. Isothermal irradiation with light of high intensity in the range 330–600 nm caused the enzyme to be reduced rapidly. Spectroscopic changes were observed which persisted after reoxidation of flavins. Intense new absorbance maxima between 310 and 330 nm together with a large decrase in absorbance at 450 nm were noted. Under controlled conditions approximately half of the total flavin and practically all of the bound FAD were labilized. NADH oxidase activity and NADH linked reduction of orotate were selectively lost. The correlation between FAD labilization and loss of activity strengthens the hypothesis that FAD represents the site of activity with NADH. Activity with NADH was partially restored by incubation of the irradiated enzyme with FAD or FMN.     

BLUE AND ULTRAVIOLET-B LIGHT PHOTORECEPTORS IN PARSLEY CELLS

Abstract— Ultraviolet-B (UV-B) and blue light photoreceptors have been shown to regulate chalcone synthase and flavonoid synthesis in parsley cell cultures. These photoreceptors have not yet been identified. In the present work, we studied UV-B photoreception with physiological experiments involving temperature shifts and examined the possible role of flavin in blue and UV-B light photoreception. Cells irradiated with UV-B light (0.5–15 min) at 2°C have the same fluence requirement for chalcone synthase and flavonoid induction as controls irradiated at 25°C. This is indicative of a purely photochemical reaction. Cells fed with riboflavin and irradiated with 6 h of UV-containing white light synthesize higher levels of chalcone synthase and flavonoid than unfed controls. This effect did not occur with blue light. These results indicate that flavin-sensitization requires excitation of flavin and the UV-B light photoreceptor. The in vivo kinetics of flavin uptake and bleaching indicate that the added flavin may act at the surface of the plasma membrane. In view of the likely role of membrane-associated flavin in photoreception, we measured in vitro flavin binding to microsomal membranes. At least one microsomal flavin binding site was solubilized by resuspension of a microsomal pellet in buffer with high KPi and NaCl concentrations and centrifugation at 38000 g. The 38000 g insoluble fraction had much greater flavin binding and contained a receptor with an apparent K_D of about 3.6 μM and an estimated in vivo concentration of at least 6.7 × 10–⁸M. Flavin mononucleotide, roseoflavin, and flavin adenine dinucleotide can compete with riboflavin for this binding site(s), although each has lower affinity than riboflavin. Most microsomal protein was solubilized by resuspension of the microsomal pellet in non-denaturing detergents and centrifugation at 38 000 g ; however, this inhibited flavin binding, presumably because of disruption of the environment of the flavin receptor. The parsley microsomal flavin binding receptor(s) have a possible role in physiological photoreception.     

Contributions of the 8-methyl group to the vibrational normal modes of flavin mononucleotide and its 5-methyl semiquinone radical

Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.     

Chromatographic determination of riboflavin and its derivatives in food

Three elution methods on two different reversed-phase C18 columns were developed to determine flavin derivatives in raw egg white, raw egg yolk, egg powder, pasteurised milk, fermented milk products and liver (chicken, calf and pig). Additionally, 11 thin-layer chromatography solvent systems were used to confirm presence of flavins detected in assessed products. It was found that an Alphabond C18 column was not as effective as a Symmetry C18 column. Method A (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0 applied on an Alphabond C18 column) can be used for determination of flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin and 10-hydroxyethylflavin in products that do not contain 7alpha-hydroxyriboflavin. Method B (mobile phase gradient of methanol-demineralized water, on an Alphabond C18 column) can be useful to separate flavin coenzymes from other flavin compounds or to confirm the presence of 7alpha-hydroxyriboflavin and 10-hydroxyethylflavin in analysed samples. Method C (mobile phase gradient of methanol-0.05 M ammonium acetate, pH 6.0, on a Symmetry C18 column) allows separation of all flavins detected in tested products: flavin adenine dinucleotide, flavin mononucleotide, riboflavin 4',5'-cyclic phosphate, riboflavin, 10-formylmethylflavin, 10-hydroxyethylflavin, 7alpha-hydroxyriboflavin, riboflavin-beta-D-galactoside and riboflavin-alpha-D-glucoside.     

KINETICS and THERMODYNAMICS OF THE AUTOXIDATION OF REDUCED FLAVIN MONONUCLEOTIDE (FMN)

Abstract— Pulse radiolysis of flavin mononucleotide (FMN) solutions produce flavin semiquinone radicals. The equilibrium constant of radical formation was determined in the pH range2–7 as a function of flavin concentration. Several complex constants as well as the kinetics of equilibration were measured in this pH regime. The rate constant of the autoxidation of the free flavin mononucleotide semiquinone radical was determined to be (1 ± 0.5) × 10⁴ M ⁻¹ s⁻¹. It is shown that between pH 2 and 7 the direct reaction of the dihydroflavin with oxygen is negligibly slow compared to the rate of autocatalysis. The autoxidation of dihydroflavin is discussed in relation to electron transfer theory.     

Raman spectra of flavins: avoidance of interference from fluorescence

Raman spectra of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in neutral aqueous solutions have been observed with excitations at 600.0, 363.8, 351.1, 337.1, and 257.3 nm. It has been suggested that, in general, an excitation in the absorption band of the second or the third longest waveleng (instead of the first) is an effective means for observing a resonance Raman spectrum of a chromophore without fluorescence disturbance.     

ACTION SPECTRA FOR PHOTOACCUMULATION OF GREEN AND COLORLESS EUGLENA: EVIDENCE FOR IDENTIFICATION OF RECEPTOR PIGMENTS

Abstract —Phototaxis action spectra have been determined in three different types of Euglena gracilis ; green and dark-bleached cells containing the stigma and streptomycin-treated white mutant with an absorptionless stigma. In all three types of microorganisms the flavin containing paraflagellar body is present. The shape of the action spectrum is the same for the three types of cells and proportional to the absorption spectrum of flavoproteins. It has been shown that the structure of the action spectrum does not depend on the presence of screening organelles, on which, instead, depends the direction of the response to the light stimulus. It is concluded that the flavin chromophores present in the photoreceptor are the pigments responsible for phototaxis in Euglena gracilis.     

BLUE LIGHT INDUCED PHOTOTRANSFORMATION OF PHYTOCHROME IN THE PRESENCE OF FLAVIN*

Abstract— Irradiation of the P_r form of phytochrome in the presence of flavin mononucleotide (FMN) which absorbs the actinic blue light yields P_fr at a rate greater than that in the absence of FMN. The actinic blue light absorbed by FMN enhances the phototransformation of P_r via the energy transfer from the former to the latter. On the other hand, the photoreversion of P_fr was inhibited by the presence of FMN when illuminated with blue light. The lack of photo-enhancement of the reversion of P_r, by blue light suggests that the P_fr chromophore (acceptor) transition dipole is virtually perpendicular to the FMN transition dipole, as the result of a chromophore reorientation in the P_r→P_fr phototransformation. The fact that blue light absorbed by flavin preferentially enhances the forward phototransformation of phytochrome while inhibiting the reversion may have an important implication in the high irradiance responses in plants in terms of a preferential accumulation of P_fr by blue light excitation.     

Bioluminophore and Flavin Mononucleotide Fluorescence Quenching of Bacterial Bioluminescence—A Theoretical Study

Bacterial bioluminescence with continuous glow has been applied to the fields of environmental toxin monitoring, drug screening, and in vivo imaging. Nonetheless, the chemical form of the bacterial bioluminophore is still a bone of contention. Flavin mononucleotide (FMN), one of the light‐emitting products, and 4a‐hydroxy‐5‐hydro flavin mononucleotide (HFOH), an intermediate of the chemical reactions, have both been assumed candidates for the light emitter because they have similar molecular structures and fluorescence wavelengths. The latter is preferred in experiments and was assigned in our previous density functional study. HFOH displays weak fluorescence in solutions, but exhibits strong bioluminescence in the bacterial luciferase. FMN shows the opposite behavior; its fluorescence is quenched when it is bound to the luciferase. This is the first example of flavin fluorescence quenching observed in bioluminescent systems and is merely an observation, both the quenching mechanism and quencher are still unclear. Based on theoretical analysis of high‐level quantum mechanics (QM), combined QM and molecular mechanics (QM/MM), and molecular dynamics (MD), this paper confirms that HFOH in its first singlet excited state is the bioluminophore of bacterial bioluminescence. More importantly, the computational results indicate that Tyr110 in the luciferase quenches the FMN fluorescence via an electron‐transfer mechanism.     

Spectroelectrochemical investigation of a flavoprotein with a flavin-modified gold electrode

A flavin-modified gold electrode was developed in order to catalyze the electrochemical oxidoreduction of flavoproteins. Surface modification was carried out by a two-step procedure. In the first step a mixed self-assembled monolayer obtained by adsorption of activated and nonactivated 3,3'-dithiopropionic acid (free acid and N-succinimidyl ester) was formed, followed by the covalent attachment of a N(10)-hexylamino-alkylated flavin derivative via an amide bond in the second step. The electrochemical properties of the flavin-modified electrode are presented and discussed. The redox potential of the attached flavin was measured at various pH values and the electron-transfer rate constant between electrode and flavin was determined as k0 = 5 s(-1) independent of pH. The flavin-modified electrode was successfully applied to the electrochemical and spectroelectrochemical investigation of the flavoprotein WrbA from Escherichia coli that shows some structural similarities to flavodoxins. It is concluded that the electron transfer "electrode --> flavin --> flavoprotein" occurs by a two-step hopping mechanism where the first step is rate determining. Kinetic details are discussed. Furthermore, it turned out that, in contrast to flavodoxins, where the semiquinone state is stabilized, WrbA rapidly takes up two electrons, directly leading to the fully reduced form. The presented electrode surface modification may generally lend itself for spectroelectrochemical investigations of flavoproteins.     

Flavin-sensitized Photo-oxidation of Lysozyme and Serum Albumin

The excited state processes of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in argon-saturated aqueous solution were studied in the presence of lysozyme or bovine serum albumin (BSA). UV–Vis absorption and fluorescence spectroscopy indicates that the noncovalent flavin-protein binding is relatively weak. Quenching of the flavin triplet state by BSA, observed by time-resolved photolysis, is less efficient than by lysozyme. Light-induced oxidation of the two proteins and reduction of the three flavins were studied. The quantum yields of the former and latter in the absence of oxygen are up to 0.1 and 0.04, respectively. The effects of pH and sensitizer and protein concentrations were examined in greater detail. The proposed reaction is electron transfer from the tryptophan moiety to the flavin triplet rather than excited singlet state.