Flavin System Research Articles

Flavin Catalysis Employing an N(5)‐Adduct: an Application in the Aerobic Organocatalytic Mitsunobu Reaction

An artificial flavin system has been firstly proved to employ an N(5)‐adduct for a catalytic transformation. This mode of catalysis occurs in some flavoenzymes but it is unknown in chemocatalysis, still exclusively using only C(4a)‐adducts. In our report, an ethylene‐bridged biomimetic flavin has been shown to participate in the Mitsunobu esterification reaction as an alternative to dialkyl azodicarboxylate. The reaction occurs via a flavin N(5)‐triphenylphosphane adduct and is catalytic from the point of view of the flavin, which is regenerated by oxygen. This approach distinguishes from other catalytic Mitsunobu reaction procedures which require an extra catalytic system.

(Invited) Limitations in Electrochemically Active Biofilms

Almost a decade has passed since the rediscovery of microbial fuel cell research, yet the power generation of these devices has not advanced. This is mainly due to the research being focused solely on the improvement of power generation rather than on a fundamental understanding of electron transfer processes and their limitations in the biofilms grown on the anode and cathode. The chemical and electrochemical gradients in these biofilms play a critical role in electron transfer rates between cells and a solid electron acceptor or donor. Our research group has developed and used novel microelectrodes and in situ Nuclear Magnetic Resonance (NMR) imaging techniques to investigate electron transfer mechanisms in biofilms. The newly developed microelectrodes were successfully operated on polarized surfaces. Both NMR and microelectrode technologies allowed us to quantify metabolic and chemical variations in biofilms while the cells respired on polarized electrode surfaces. We combined our techniques with two new techniques – rotating disk electrode (RDE) and electrochemical quartz crystal balance (eQCM). The addition of both these techniques allowed us to differentiate mass transfer limitations from intra-biofilm electron transfer processes. When we attempted to identify limitations in anodic biofilms, first, we addressed the importance of local acetate concentrations within G. sulfurreducens biofilms using a novel acetate microelectrode which has better resolution than NMR. The microelectrode work confirmed our previous NMR findings and generated profiles on the order of µM. We should note that the microelectrode method developed is significantly more economical to use than NMR and can facilitate rapid in situ measurements of acetate in anoxic biofilms. Second, we integrated our previous RDE method with our eQCM methods to show that, on both a per current basis and a per mass basis, biofilm capacitance is a better indicator of biofilm performance than conductance.In particular, we compared the capacitance/conductance relationship of G. sulfurreducens biofilms to those of both polyaniline films and diffusing flavin systems. The results indicated that the tandem increase in capacitance and conductance is unique to the extracellular electron transfer mechanisms operating in G. sulfurreducens biofilms. Our local potential and current measurements indicated that the upper layers of the biofilms are always under a reducing condition. This knowledge also confirmed that the amount of cytochromes in the biofilm is not high enough or the biofilm conductivity is not sufficient to generate an oxidizing condition near the top of a biofilm. Finally, we extended the role of excess surface area in the scale-up of bioelectrochemical systems by examining biomass accumulation under this condition. We found that biomass continues to accumulate on the excess surface area electrodes with net arithmetic growth rates and acetate consumption during steady-state current. The linear growth rates constrained by ion transport limitations that we found may be important factors in understanding the current generation using high-surface-area 3D electrodes with advection. In conclusion, we demonstrated the importance of the metabolic inactivity of layers within G. sulfurreducens biofilms using microelectrodes and NMR. In addition, we verified these findings using electrochemical impedance spectroscopy (EIS) by demonstrating the pseudocapacitive behavior of biofilm impedance. We also demonstrated the feasibility of directly measuring biofilm inefficiencies via intra-biofilm electron transfer rates and local current depth profiles.

Artificial Flavin Systems for Chemoselective and Stereoselective Oxidations

AbstractFlavinium salts, both isoalloxazinium and alloxazinium derivatives, are useful organocatalysts of redox reactions, and particularly in recent years, the number of transformations catalysed by flavinium salts has increased. This review outlines the synthetic applications of flavinium catalysts in oxygenation reactions with oxygen and hydrogen peroxide as terminal oxidising agents; these oxidations involve flavin hydroperoxide as an intermediate, thus mimicking flavin monooxygenases. Special attention is paid to mechanistic studies and to the design of catalytic systems, especially the effects of the structures of flavinium catalysts, and reaction conditions on the efficiency, chemoselectivity and stereoselectivity of oxygenations.

Effect of intramolecular hydrogen bonding on biomimetic reactions by flavins

Abstract Selected 6- and 7-carboxyflavins activate H2O2, oxidizing thioanisole to its sulfoxide. The greater reactivity of the 6-carboxyflavin derivatives are ascribed to the intramolecular hydrogen bonding between N(5) and the carboxyl group of the flavin system.

Cover Picture: Small 11/2008

The cover picture shows scanning electron microscopy images of organic nanowires and platelets. High-aspect- ratio nanowires were formed via synergistic hydrogen bonding and dipole–dipole interactions of a hydrogen-bonding azobenzene flavin system. In contrast, micrometer-size platelets were formed using a highly analogous non-hydrogen-bonding molecule. Co-precipitation of the two analogous molecules provides efficient control over both nanowire length and diameter. This strategy offers an opportunity to engineer supramolecularassemblies with well-defined size, shape, and morphology for electronic and optical applications. For more information, please read the Full Paper “Controlled Self-Assembly of Organic Nanowires and Platelets using Dipolar and Hydrogen Bonding Interactions” by G. Cooke, V. M. Rotello, et al. beginning on page 2074.

Crystal Structure of a Minimal Nitroreductase, ydjA, from Escherichia coli K12 with and without FMN Cofactor

Nitroreductases (NTR) are enzymes that reduce hazardous nitroaromatic compounds and are of special interest due to their potential use in bioremediation and their activation of prodrugs in directed anticancer therapies. We elucidated the crystal structures of ydjA from Escherichia coli (Ec_ydjA), one of the smallest NTRs, in its flavin mononucleotide (FMN)-bound and cofactor-free forms. The α + β mixed monomeric Ec_ydjA forms a homodimeric structure through the interactions of the long central helices and the extended regions at both termini. Two FMN molecules are bound at the dimeric interface. The absence of the 30 internal amino acids in Ec_ydjA, which forms two helices and restricts the cofactor and substrate binding in other NTR family members, creates a wider and more flexible active site. Unlike the bent FMN ring structures present in most NTR complexes currently known, the flavin system in the Ec_ydjA structure maintains a flat ring conformation, which is sandwiched between a Trp and a His residue from each monomer. The analysis of our Ec_ydjA structure explains its specificity for larger substrates and provides structural information for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

Structural insights into the mechanism of amine oxidation by monoamine oxidases A and B

Due to their pharmacological importance in the oxidation of amine neurotransmitters, the membrane-bound flavoenzymes monoamine oxidase A and monoamine oxidase B have attracted numerous investigations and, as a result, two different mechanisms; the single electron transfer and the polar nucleophilic mechanisms, have been proposed to describe their catalytic mechanisms. This review compiles the recently available structural data on both enzymes with available mechanistic data as well as current NMR data on flavin systems to provide an integration of the approaches. These conclusions support the proposal that a polar nucleophilic mechanism for amine oxidation is the most consistent mechanistic scheme as compared with the single electron transfer mechanism.

π…︁π interactions of flavins, 5. Syntheses, structures and physical properties of flavin systems with covalent bonding to π donors and π acceptors (quinones)

AbstractTo study π…︁π interactions of flavins of different redox states with π‐electron donors and acceptors, the syntheses of compounds in which such π systems are linked to flavins by using the „cyclophane concept”︁ were attempted and partially achieved. The syntheses of the metacyclo‐isoalloxazinophanes 1 and 2 are reported as well as the X‐ray structure analysis of 2. The conversion of 1 or 2 to the benzoquinono‐isoalloxazinophanes 3 or 4, respectively, was not achieved, obviously due to the specific sterical shielding of the methoxy groups in the isoalloxazinophanes. Benzoquinone‐isoalloxazine systems in which the two components are linked by only one triethylene chain were obtained. Spectroscopic and electrochemical properties related to intramolecular interactions are reported.

Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb(3+)) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb(3+) to oxyferrous (Lb(2+) · O2) or deoxyferrous (Lb(2+)) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O2 the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb(3+) reduced) than in the presence of O2; this phenomenon was probably because O2 competes with Lb(3+) for reductant, thus generating activated O2 species. The flavin-mediated reduction of Lb(3+) did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb(3+) directly. The NAD(P)H + flavin system also reduced the complexes Lb(3+) · nicotinate and Lb(3+) · acetate to Lb(2+) · O2, Lb(2+) or Lb(2+) · nicotinate, depending on the concentrations of ligands and of O2. In the presence of 200 μM nitrite most Lb remained as Lb(3+) in aerobic conditions but the nitrosyl complex (Lb(2+) · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb(3+) at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb(3+) in vivo.

Further consideration of flavin coenzyme biochemistry afforded by geometry-optimized molecular orbital calculations.

Investigation of lumiflavin and several other isoalloxazine ring derivatives has been carried out by geometry-optimized molecular orbital calculations. The results have provided insight into the flexibility of the flavin cofactor in the reduced and oxidized states, the regions of the fused three-ring system that should play an important role in flavin electron transfers, and the structural and functional role of the xylene and heteroatomic portions of the flavin system. The significance of these results is reviewed in relation to the experimentally identified chemical and biochemical properties of the flavin nucleotide coenzymes.

Electron-spin-resonance studies on flavoenzymes.

Our current knowledge on the spin distribution of flavoenzyme semiquinones would suggest that it is very similar to that determined for model flavin systems. It remains for future work to establish whether this conclusion is valid for a wide range of flavoenzymes as well as for those in which the flavin has become covalently attached to the protein via the 8 alpha position. The improvement in e.s.r. instrumental capabilities, advances in ENDOR spectroscopy, and the application of other techniques such as electron spin echo spectroscopy should result in considerable new information regarding the structures and environment of flavoenzyme semiquinones. Further application of these physical approaches to flavoenzymes in which chemically modified flavins have been substituted for the 'normal' coenzyme (Massey & Hemmerich, 1980) may be particularly informative.

ROLE OF COFACTORS IN BREAKDOWN OF TMAO IN FROZEN RED HAKE MUSCLE

Previously indicated results that cofactor concentrations were the rate-limiting factor in the production of DMA in frozen red hake muscle were confirmed. Both the Fe-reducing agent (ascorbate, cysteine) and the flavin-NADH systems which had been previously shown to be effective in vitro, were demonstrated to also function in minced muscle and in reconstituted muscle, i.e., muscle from which the low molecular weight fraction had been removed. The evidence implies that an oxidation-reduction cycle of Fe is involved in the breakdown of TMAO to DMA. Activity with the flavin system is greatly increased in the presence of glucose oxidase and glucose which would remove O2 from the system. Fe was effective in increasing DMA production under all conditions, whereas Fe+3 was effective only in the presence of reducing agents and/or anaerobic conditions. Enzymes capable of destroying cofactors when added to minced muscle tissue before frozen storage inhibited the rate of formation of DMA. The percentage of formaldehyde produced which was bound (unreactive to the Nash reagent) was much higher in minced muscle than in reconstituted muscle, indicating that a large fraction of the formaldehyde produced reacts with the low molecular weight fraction.

Structure and properties of 5-deazaflavin radicals as compared to natural flavosemiquinones

In order to gain more insight into flavin radicals, on which the selection of 1 e −- and 2 e −-oxireduction modes in flavoproteins depends, we have investigated structure, spectral properties and decay mode of molecular species occurring in the half-reduced 5-deazaflavin ‘model’ system by flash photolysis and pulse radiolysis. 1. (1) Enforced 1 e −-reduction of 5-deazaflavin yields the short-lived red-colored 1-HdḞl, which is a strong reductant. In the absence of any electron acceptor, this radical decays by 1,5-prototropy (see below) and dismutation, which is rapidly reversed upon illumination. Competing with this photo-comproportionation, irreversible formation of the photo-stable σ-dimer ( Hd F ̇ l ) 2 , linked via C(5), is observed, which becomes prevalent under prolonged illumination. 2. (2) Enforced 1 e −-abstraction from 1,5-dihydro-5-deazaflavin yields the tautomeric 5-HdḞl, which is a mild oxidant and is transparent at λ > 480 nm. Prototropy 5- Hd F ̇ l ⇆ 1- Hd F ̇ l can be rate-determining in 5-deazaflavin redox reactions. Hence, the radical state in the 5-deazaflavin system does not mediate double 1 e −-oxidoreduction as do natural flavosemiquinones. Instead, 5-deazaflavin favors nucleophilic substrate addition (carbanion transfer) and formation of intermediate σ-adducts in (photo)reductions even over the extent obsersed with natural flavin. This confirms the description of 5-deazaflavin as a ‘flavin-shaped nicotinamide derivative’. It explains at the same time the mechanism of 5-deazaflavin acting as a mild and yet potent photosensitizer in 1 e −-reductions of biological redox systems. 3. (3) It is shown that replacement of N(5) by CH in the flavin nucleus also leads to the disappearance of the known action-p K in the photoreduction, which confirms the assignment of the latter p K in the natural flavin system to 5-protonation of the excited flavin triplet. From these model studies the following biological conclusions can be confirmed: The tautomer equilibrium of natural flavin semiquinones is diffusion-controlled and regulated thermodynamically: 5 H F ̇ l ⇄ 1- H F ̇ l , while in flavoproteins the same equilibrium is regulated by regiospecific H-bridges from the apoprotein, which thus decides between 1 e −- (stable 5- H F ̇ l ) and 2 e −-reaction (unstable 1-HḞl) modes.

5‐Thia‐5‐Deazaflavin, a 1e−‐Transferring Flavin Analog

By sulfur substitution of the N‐5 atom in flavins and flavocoenzymes a flavin analog is obtained, 5‐thiaflavin, which is found to be isoelectronic and isosteric with natural flavin in the fully reduced and half‐reduced states, but not in the oxidized state. Among the three ‘redox shuttles’ characterizing the flavin system, viz. upper 1e−, lower 1e− and 2e− shuttle, only the second one is retained in thiaflavin, which limits the redox activity of this system to 1e− transfer.The structure and properties of the molecular species participating in the thiaflavin redox system are discussed in comparison with the flavin system. The corresponding chemistry of a ‘2e− flavin’, 5‐deazaflavin, has been treated in the preceding paper.5‐Thiaflavin is found to exhibit a stable neutral radical, which is analogous to the ‘blue’ flavosemiquinone. Unlike normal flavin, where the radical is in a dismutation equilibrium, thiaflavin radical shows reversible formation of a covalent dimer, which is stable in aprotic solution and disproportionates only in water, with irreversible formation of a sulfoxide. The ultraviolet and infrared spectra of the dimer are in agreement with the structure of two 5‐thiaflavin molecules linked covalently at their 4a carbons. This corroborates the earlier hypothesis that the essential intermediate in the dismutation of normal flavin is likewise a covalent dimer.Thiaflavin is tightly bound by apoflavodoxin. The protein catalyses the autoxidation to the radical state. Thiaflavodoxin radical is even more stable (towards further oxidation) than is the free thiaflavin radical.The redox potential of the couple reduced thiaflavin/thiaflavin radical (sFlred/sFl) is surprisingly high. From the reversible equilibrium established with ferricyanide, sFlred+ Fe(CN)63−⇄ sFl·+ Fe(CN)64−, the standard potential of the sFlred/sFl· couple, Em at pH 7, has been estimated as + 0.38 V.

Nucleophilic addition reactions of free and enzyme-bound deazaflavin.

DeazaFMN-containing glycolate oxidase has been prepared and shown to catalyze the stereospecific transfer of the alpha-hydrogen from substrate to enzyme-bound deazaFMN. The reaction of sulfite, cyanide, and hydroxylamine with several deazaflavin-containing enzymes (glycolate oxidase, D-amino acid oxidase, glucose oxidase, N-methylglutamate synthetase) and free deazaFMN has been examined. All the deazaflavin systems tested form reversible 1:1 complexes with sulfite and cyanide. The pH dependence of the reaction of free deazaFMN with cyanide indicates that cyanide anion is the reacting nucleophile. Hydroxylamine complexes are formed with deazaFMN glycolate oxidase and deazaFAD glucose oxidase. The effectiveness of the various nucleophilic reagents in complex formation decreases in the following order: sulfite greater than cyanide greater than hydroxylamine. The relative stability observed for the sulfite and cyanide complexes formed with various deazaflavin systems (glycolate oxidase greater than D-amino acid oxidase greater than free deazaFMN) follows the same trend observed for the stability of the sulfite complexes formed with the corresponding flavin system. A correlation is also observed between the reduction potential (E'o) of the deazaflavin system (glycolate oxidase (- 170 mV) greater than D-amino acid oxidase (-240 mV) greater than free deazaFMN (-178 mV) and the stability of the deazaflavin-nucleophile complexes. The following evidence indicates that deazaflavin systems are generally more susceptible toward nucleophilic attack than corresponding flavin system: (a) with the exception of glucose oxidase, the dissociation constants for the deazaflavin-sulfite complexes are at least 1 order of magnitude less than the corresponding flavin sulfite complexes; (b) the least reactive nucleophile, hydroxylamine, does not form a complex with any of the flavin systems. In the case of cyanide, a complex is formed only with native glycolate oxidase, which is the flavin-containing system most susceptible to attack by the more reactive sulfite. Formation of the various (deaza)flavin-nucleophile complexes is characterized by a bleaching of the longer wavelength absorption band of the chromophore and increases in absorption below the isosbestic point of the reaction in the near-ultraviolet region of the spectrum. These results are consistent with the formation of covalent adducts via attack of the various nucleophiles at position 5 of (deaza)flavin. The reaction with cyanide provides the first example of a reversible addition of carbanion to enzyme-bound (deaza)flavin.

Cytochromes in Streptococcus faecalis var. zymogenes grown in a haematin-containing medium.

Summary: Functional cytochromes were found in the membrane fraction of Streptococcus faecais var. zymogenes grown aerobically with haematin. Molar growth yields shovei that the cytochrome system produced additional ATP. Inhibitors and uneruglers of oxidative phosphorylation verified the presence of cytochromes in the membrane fraction. Spectra indicated both a and b type cytochromes when cultures were grown with haematin. Without haematin, only a flavin system of electron imsport developed without additional ATP generation. Bacteria grown anaeuoically with haematin did not form cytochromes.

ROLE OF CHARGE‐TRANSFER INTERACTIONS IN FLAVOPROTEIN CATALYSIS

The last decade has produced a veritable explosion of knowledge on the chemical reactivity of the isoalloxazine ring system of the flavin coenzymes, largely because of a side-by-side development with knowledge of flavoenzymes and model flavin studies. In this period the visible absorption spectra and epr spectra of the flavin semiquinone in its cationic, neutral, and anionic forms have been defined'- (FIGURE 1 ) . In the same period a large number of flavoenzymes have been investigated by rapid reaction spectrophotometric techniques, and in nearly all cases distinctive spectra characterized by longwavelength-absorption bands have been detected as catalytic intermediates. With few exceptions, the spectra of the intermediates are unequivocally different from those of flavin semiquinone in either its neutral or its anionic form. As will be discussed in more detail below, these intermediates are in general readily thought to contain the flavin in the oxidized or reduced state, on the basis of their spectral properties. During this period the great chemical reactivity of the flavin system has been uncovered.',8 This model work has led to the synthesis of well-defined substituted isoalloxazines, with substituents at almost every atom of the isoalloxazine ring system. The recognition of this great reactivity has led to the obvious suggestion that intermediates in flavoenzyme catalysis involve covalent linkage of substrate moieties with the flavin.'-'l While the formation of such intermediates cannot be excluded rigorously on the basis of present evidence, the known spectral properties of such model substituted flavins fail to account for the intermediates observed in flavoenzyme catalysis.

Studies on Oxidation-Reduction Potentials (ORP) of Microbial Cultures Part IV

Mycelia of Rhizopus G-36 after 36 hr-culture produced lactic acid even in the absence of oxygen. But unexpectedly, the ORP under anaerobic secondary culture was exactly the same as that in the aerobic shaking culture (rH 13.2). A method for homogenization of the culture without secondary oxidation was improved. The ORP of anaerobically homogenized cultures was rH 11, and was thought to be due to the activities of all redox systems in the mycelium. The respiration system of this strain was switched from cytochrome system to flavin system at the point of change in KCN-sensitivity. The ORP of this strain may be influenced by respiration through the flavin system.

Progress in the chemistry and molecular biology of flavins and flavocoenzymes.

AbstractAn attempt is made to create a basis for the understanding of flavin‐ (Vitamin B2‐) dependent oxidations in terms of molecular structure. The main points of this survey can be summarized as follows: (a) The flavins must be differentiated according to their redox states (quinone state, radical state, hydroquinone state), since configuration, energy content of the tautomeric forms, metal affinity etc., alter drastically during reduction. – (b) The methyl group on C‐8 of the flavoquinone is reactive. – (c) The electron transfer properties of the flavin system frequently depend on the coupling of secondary redox systems to the apoprotein. This interaction thermodynamically stabilizes the half‐reduced state of the flavin. – (d) Numerous novel flavin types, among them stabilized free radicals, have become available through syntheses at the dihydro stage. – (e) Reaction of the flavin radical with O2 is slow as long as no disproportionation occurs, i.e. the fast flavin autoxidation proceeds via the dihydro state. – (f) The flavin nucleus gains affinity for heavy metal ions only by the acceptance of one, and only one, electron. – (g) Redox activity and contact with the flavin coenzyme have been confirmed for Fe and Mo, which occur in flavoproteins. The properties of the metal coordination sphere are determined primarily by sulfur‐containing groups of the apoprotein.

Synthesis in vitro of thyroxine from diiodotyrosine by myeloperoxidase and by a cell-free preparation of beef thyroid glands II. Flavin mononucleotide and Mn2+ system

1.1. Thyroxine was identified by paper chromatography as one of the products of the action of a purified peroxidase, myeloperoxidase, on diiodotyrosine in the presence of FMN, MnCl2 and light as a hydrogen peroxide-generating system.2.2. The incubation of diiodotyrosine with a cell-free thyroid particulate preparation under comparable conditions also led to the formation of thyroxine which was identified by paper chromatography, ultraviolet and infrared analyses and by the stimulation of tadpole metamorphosis.3.3. The synthesis of thyroxine was inhibited by ergothioneine, reduced gluthione, ascorbic acid, semicarbazide, Tapazole, 2-thiouracil and catalase (EC 1.11.1.6).4.4. Significant amounts of ammonia were released on the incubation of diiodotyrosine with either myeloperoxidase or the thyroid particulate fraction in the presence of FMN, MnCl2 and light. Ammonia release was inhibited by the same reagents that inhibited thyroxine synthesis.5.5. The synthesis of thyroxine in the flavin system differed from the synthesis in the glucose and glucose oxidase system1 in several respects: heat inactivation; specificity for the thyroid particulate fraction; effect of serotonin.6.6. These results are discussed in relation to the possible involvement of a non-specific protein “activation”, in addition to a thyroid peroxidase, in the synthesis of thyroxine from diiodotyrosine in the presence of FMN, MnCl2 and light.7.7. The possibility that there existed a peroxidase catalyzed transiodination of iodide from diiodotyrosine to endogenous free thyronines was entertained. Under the experimental conditions employed in this study, such a reaction is considered unlikely.