REACTIVITY OF EXCITED STATES OF FLAVIN AND 5-DEAZAFLAVIN IN ELECTRON TRANSFER REACTIONS

Abstract— Quenching of the excited states of lumiflavin and 3-methyl-5-deazalumiflavin by methyl-and methoxy-substituted benzenes and naphthalenes in methanol was investigated. The observed difference in the reactivity of acid and neutral lumiflavin triplets is explained thermodynamically by applying the Michaelis cycle, as being due to the higher reduction potential of the acid triplet. In this connection the p K values of lumiflavin triplet (p K ^M= 6.5) and semiquinone (p K ^M= 11.3) have also been determined in methanol. The difference in the reactivity between the singlet and triplet states of lumiflavin is found to be greater as predicted by the difference in excitation energy. The reactivities of the excited states of flavin and 5-deazaflavin differ only slightly in contrast to the marked difference in the ground state reactivities of electron transfer reactions. This is explained in terms of the model of Rehm and Weller. The pH dependence of the electron transfer quenching of 5-deazaflavin triplet was investigated in water, yielding a triplet p K of 2.5. In contrast to the flavin, this triplet p K does not significantly differ from the p K of the 5-deazaflavin ground state. From this, different sites of protonation are deduced for the photoexcited triplet states of flavin and 5-deazaflavin.     

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.     

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.     

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 COMPOUNDS AS AGENTS FOR THE OXIDATION OF PLASTOCYANIN IN BLUE LIGHT

Abstract—Flavins, flavin nucleotides and selected flavoproteins have been compared in a reaction using blue light. in which plastocyanin is oxidized as the flavin is photoreduced. Per unit of light absorbed. flavin mononucleotide is more effective than flavin adenine dinucleotide, ribotlavin or lumiflavin. Of the flavoproteins tested, diaphorase from Clostridium kluyveri was most effective, but was less active than free flavin mononucleotide. The oxidation of plastocyanin requires aerobic conditions. and appears to be mediated by the production of singlet oxygen when the flavin is irradiated.     

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.     

Flavoenzymes that catalyse reactions with no net redox change

This review covers unusual flavoenzymes that catalyse reactions with no net redox change. Some of these enzymes utilise the redox properties of flavin directly in catalysis with either two-electron chemistry (N-methylglutamate synthase and 5-hydroxyvaleryl-CoA dehydratase) or free radical chemistry (chorismate synthase, DNA photolyase, (6-4) photolyase and 4-hydroxybutyryl-CoA dehydratase). Whether the flavin has a redox role in some other flavoproteins is not yet clear ((R)-2-hydroxyacyl-CoA dehydratases, isopentenyl diphosphate isomerase and UDPgalactopyranose mutase). The remaining flavoenzymes do not make use of the redox properties of the flavin (acetohydroxyacid synthases and hydroxynitrile lyase). The literature is reviewed up to early 2002 and 121 references are cited.     

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.     

On mechanistic aspects of 5-deazaflavin dependent dehydrogenation of alcohol

The reaction of 5-deazaflavins with alcoholates was investigated and the direct hydride equivalent transfer from C₁ of alcoholates to C₅ of 5-deazaflavins was confirmed by chemical methods. 5-Alkoxy-10-butyl-3-methyl-5-deazaflavins were synthesized by treatment of 10-butyl-5-chloro-3-methyl-5-deazaflavin with the corresponding alcoholates. The 5-alkoxy-5-deazaflavins were reduced by sodium borodeuteride or sodium hydro-sulfite in deuterium oxide or monodeuteriomethanol to give 10-butyl-3-methyl-1,5-dihydro-5-deazaflavin-5,5-D₂ exclusively. 3,10-Dimethyl-5-deazaflavin radical anion was detected by esr technique on treatment of 3,10-dimethyl-5-deazaflavin with potassium in DMF. From the above reactions, a mechanism of 5-deazaflavin dependent dehydrogenation of alcoholate was proposed.     

The photoreduction of 8-substituted 5-deazaflavins and the 5-deazaflavin catalyzed photoreduction of methyl viologen

Prolonged illumination of 8-X-5-deazaflavins (X = C1, N(CH₃)₂, NH₂, p-NH₂-C₆H₄) in the presence of an electron donor leads to the formation of a 5,5′-dimer and/or a 6,7-dihydro compound. The course and rate of these photoreductions were established and discussed in terms of electronic and steric effects, exerted by the substituent at position 8 and the electron donor. Pseudo first-order kinetics were found to apply to the photoreduction of 8-X-5-deazaflavins (X = Cl, NH₂, p-NH₂-C₆H₄) while the rate of the photoreduction of 8-X-5-deazaflavin (X = N(CH₃)₂) appeared to contain an autocatalytic element. The catalytic effect of 8-X-5-deazaflavins in the photoreduction of methyl viologen by EDTA was investigated. The substituent effect on the rate of the 8-X-5-deazaflavin mediated photoreduction of methyl viologen by EDTA was found to be comparable with that on the photoreduction rate of 8-X-5-deazaflavin in the presence of EDTA with the exception of 8-X-5-deazaflavin (X = N(CH₃)₂), which showed a remarkable relative enhancement of the reactivity towards methyl viologen photoreduction.     

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.     

FLUORESCENCE QUENCHING FOR FLAVINS INTERACTING WITH EGG WHITE RIBOFLAVIN-BINDING PROTEIN

Abstract— The effect of flavin structure variation upon the binding process of flavin to hen egg white riboflavin was studied using fluorescence methods for formylmethylflavin (FMF), riboflavin (RF) and flavomononucleotide (FMN).
Measurements of flavin fluorescence intensities (steady state and phase-sensitive) and lifetimes were performed in a variety of RBP concentrations and temperatures (4 to 40°C). No fluorescence of flavoproteins was detected, while the fluorescence of flavins was found to be quenched by RBP. The overall quenching process is dominated by the static quenching (about 88%) due to the flavoprotein complex formation in the ground state, presumably a charge transfer complex.     

A FLAVOPROTEIN IN Phycomyces blakesleeanus WITH SHORT FLUORESCENCE LIFETIME

Abstract— The photoreceptor system for the blue-light-induced phototropism in the fungus Phycomyces blakesleeanus includes one or more flavin chromophores, probably located in the plasma membrane of the sporangiophore. From a plasma-membrane fraction of short synchronous sporangiophores, we have enriched, by column chromatography, specific proteins with covalently bound flavins. These flavoproteins were analyzed by a fluorescence lifetime assay. Flavoproteins with fluorescence lifetimes significantly less than 5 ns have been predicted to be involved in blue-light reception. We studied samples from a wild type strain and from the night-blind mutant LI. (genotype madC). For the enriched flavoprotein samples, we found predominant fluorescent components with lifetimes of 1.5 ns (74%) for wild type and 1.8 ns (83%) for LI. According to this assay, one or both of these flavoproteins are likely components of the blue-light photoreceptor system in P. blakesleeanus.     

Structural and Functional Analysis of a Prokaryotic (6-4) Photolyase from the Aquatic Pathogen Vibrio Cholerae†

Photolyases are flavoproteins, which are able to repair UV-induced DNA lesions in a light-dependent manner. According to their substrate, they can be distinguished as CPD- and (6-4) photolyases. While CPD-photolyases repair the predominantly occurring cyclobutane pyrimidine dimer lesion, (6-4) photolyases catalyze the repair of the less prominent (6-4) photoproduct. The subgroup of prokaryotic (6-4) photolyases/FeS-BCP is one of the most ancient types of flavoproteins in the ubiquitously occurring photolyase & cryptochrome superfamily (PCSf). In contrast to canonical photolyases, prokaryotic (6-4) photolyases possess a few particular characteristics, including a lumazine derivative as antenna chromophore besides the catalytically essential flavin adenine dinucleotide as well as an elongated linker region between the N-terminal α/β-domain and the C-terminal all-α-helical domain. Furthermore, they can harbor an additional short subdomain, located at the C-terminus, with a binding site for a [4Fe-4S] cluster. So far, two crystal structures of prokaryotic (6-4) photolyases have been reported. Within this study, we present the high-resolution structure of the prokaryotic (6-4) photolyase from Vibrio cholerae and its spectroscopic characterization in terms of in vitro photoreduction and DNA-repair activity.     

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.     

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.     

Advances in the chemistry of natural flavins

The review discusses advances in the chemistry of isoalloxazines in the last 10 years, including 1978, in the field of the detection of new derivatives of flavin (7,8-dimethylisoalloxazine) and modifications of it in natural sources — microorganisms, fungi, and plant and animal tissues — and their isolation from these sources. Questions of the establishment of structure, chemical and physicochemical properties, chemical synthesis, and the biological activity of the new flavin vitamins and prosthetic groups of flavoproteins are considered. The priority of Soviet scientists in the discovery of the covalent type of bond with the prosthetic group in some flavin enzymes is noted.All-Union Scientific-Research Vitamin Institute, Moscow. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 255–275, May–June, 1979.     

Relationship between charge-transfer interactions, redox potentials, and catalysis for different forms of the flavoprotein component of p-cresol methylhydroxylase

Thirty-three variants of the flavoprotein component of p-cresol methylhydroxylase that contain noncovalently or covalently bound flavin adenine dinucleotide (FAD) analogues were studied. A very good correlation was found between the efficiency of p-cresol oxidation by these proteins and E(CT), the energy for the maximum wavelength for the charge-transfer band of the complex between the bound flavin and 4-bromophenol, a substrate mimic. The correlation covers a range of k(cat) values that spans over 5 orders of magnitude and values of E(CT) that span 900 mV, and the analysis of the data provided a value of the transfer coefficient, alpha, of 0.31. This study demonstrates clearly that the redox properties of both the bound substrate and the flavin cofactor must be taken into account to explain the relative catalytic efficiencies of the variant flavoproteins.     

FLUORESCENCE PROPERTIES OF ISOTROPICALLY AND ANISOTROPICALLY EMBEDDED FLAVINS

Abstract— On the basis of corrected fluorescence excitation and emission spectra, flavin photo-processes in anisotropically vesicle-bound flavins have been studied. By means of aliphatic C₁₈H₃₇-chains at positions 3, 7 and 10, the flavin nucleus can be variously anchored within the membrane/water interface (amphiflavin), thereby mimicking the various positions and microenvironments of the isoalloxazine ring in flavoproteins. From polarization spectra, the angles between the different electronic transition moments of isotropically dissolved or membrane-bound flavins have been obtained. Polarization and angle spectra of isotropically and various anisotropically embedded flavins exhibit strong differences, reflecting the specific interaction with the matrices. Based on a slightly modified theory, originally developed by Perrin and Weber, using the concentration dependence of fluorescence polarization, it is found that the radiative flavin-flavin interaction (selfcontact) on the membrane is by a factor of 25 to 54 smaller than in isotropic solution. This is taken as further justification to study anisotropic flavin chemistry on the basis of flavin-loaded vesicles.     

Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.