Presence Of Flavins Research Articles

Cover Picture: Materials and Corrosion. 06/2021

Cover:The effect of the presence of redox active molecules during a mild anodic polarization of AISI 304 stainless steels was studied with a focus on corrosion and microbially influenced corrosion (MIC) processes. The two‐part publication demonstrates that the presence of minute amounts of flavins in the preconditioning medium is capable of altering the pit nucleation processes (Part I) and promotes subsequent biofilm formation (Part II). Unexpectedly, biofilms formed on surfaces preconditioned in the presence of flavins led to corrosion inhibition.More detailed information can be found in:Nina Wurzler, Oded Sobol, Korinna Altmann, Jörg Radnik, Ozlem Ozcan, Preconditioning of AISI 304 stainless steel surfaces in the presence of flavins – Part I: Effect on surface chemistry and corrosion behavior, Materials and Corrosion 2021, 72, 974.and Nina Wurzler, Jan D. Schutter, Ralph Wagner, Matthias Dimper, Vasile‐Dan Hodoroaba, Dirk Lützenkirchen‐Hecht, Ozlem Ozcan, Preconditioning of AISI 304 stainless steel surfaces in the presence of flavins – Part II: Effect on biofilm formation and microbially influenced corrosion processes, Materials and Corrosion 2021, 72, 983.

Dissimilatory Fe(III) Reduction Controls on Arsenic Mobilization: A Combined Biogeochemical and NanoSIMS Imaging Approach.

Microbial metabolism plays a key role in controlling the fate of toxic groundwater contaminants, such as arsenic. Dissimilatory metal reduction catalyzed by subsurface bacteria can facilitate the mobilization of arsenic via the reductive dissolution of As(V)-bearing Fe(III) mineral assemblages. The mobility of liberated As(V) can then be amplified via reduction to the more soluble As(III) by As(V)-respiring bacteria. This investigation focused on the reductive dissolution of As(V) sorbed onto Fe(III)-(oxyhydr)oxide by model Fe(III)- and As(V)-reducing bacteria, to elucidate the mechanisms underpinning these processes at the single-cell scale. Axenic cultures of Shewanella sp. ANA-3 wild-type (WT) cells [able to respire both Fe(III) and As(V)] were grown using 13C-labeled lactate on an arsenical Fe(III)-(oxyhydr)oxide thin film, and after colonization, the distribution of Fe and As in the solid phase was assessed using nanoscale secondary ion mass spectrometry (NanoSIMS), complemented with aqueous geochemistry analyses. Parallel experiments were conducted using an arrA mutant, able to respire Fe(III) but not As(V). NanoSIMS imaging showed that most metabolically active cells were not in direct contact with the Fe(III) mineral. Flavins were released by both strains, suggesting that these cell-secreted electron shuttles mediated extracellular Fe(III)-(oxyhydr)oxide reduction, but did not facilitate extracellular As(V) reduction, demonstrated by the presence of flavins yet lack of As(III) in the supernatants of the arrA deletion mutant strain. 3D reconstructions of NanoSIMS depth-profiled single cells revealed that As and Fe were associated with the cell surface in the WT cells, whereas for the arrA mutant, only Fe was associated with the biomass. These data were consistent with Shewanella sp. ANA-3 respiring As(V) in a multistep process; first, the reductive dissolution of the Fe(III) mineral released As(V), and once in solution, As(V) was respired by the cells to As(III). As well as highlighting Fe(III) reduction as the primary release mechanism for arsenic, our data also identified unexpected cellular As(III) retention mechanisms that require further investigation.

Extracellular Electron Shuttling Mediated by Soluble c-Type Cytochromes Produced by Shewanella oneidensis MR-1.

How metal-reducing bacteria transfer electrons during dissimilatory energy generation under electron acceptor-limited conditions is poorly understood. Here, we incubated the iron and manganese-reducing bacterium Shewanella oneidensis MR-1 without electron acceptors. Removal of soluble extracellular organic compounds (EOCs) dramatically retarded transfer of electrons to an experimental electron acceptor, Cr(VI), by MR-1. However, the return of either high MW (>3000 Da) or low MW (<3000 Da) soluble EOCs produced by MR-1 to washed cells restored Cr(VI) reduction though Cr(VI) reduction was fastest when both size fractions were added together. Spectral and electrochemical characterization of EOCs indicated the presence of flavins and c-type cytochromes (c-Cyts). A model of the kinetics of individual elementary reactions between cells, flavins, released c-Cyts, and Cr(VI), including the direct reduction of flavins, released c-Cyts, and Cr(VI) by cells and the indirect reduction of Cr(VI) by reduced forms of flavins and released c-Cyts, was developed. Model results suggest that released c-Cyts could act as electron mediators to accelerate electron transfer from cells to Cr(VI), and the relative contribution of this pathway was higher than that mediated by flavins. Hence, extracellular c-Cyts produced by MR-1 likely play a role in extracellular electron transfer under electron acceptor-limited conditions. These findings provide new insights into extracellular electron shuttling and the metabolic strategy of metal-reducing bacteria under electron acceptor-limited conditions.

EPS bound flavins driven mediated electron transfer in thermophilic Geobacillus sp.

Through extracellular electron transfer (EET), bacteria are capable of transforming different insoluble materials of geochemical interest into energy-rich molecules for their growth. For this process, bacteria have been depending directly or indirectly on molecules synthesized within the cells or by various synthetics as mediators. Herein, we studied the in-situ change in electrochemistry and supporting components for EET in the extracellular polysaccharide (EPS) producing biofilm of thermophilic Geobacillus sp. The CV and DPV resultsrevealed that the intact biofilm of bacteria was not able to generate any potential at 25 °C /- ≤50 °C. However, at 55 °C (optimal condition), the potential occurred drastically after the EPS production by bacteria. HPLC and MALDI-TOF results revealed that the presence of Flavins, which can able adsorbed to the electrodes from the cell surface. Moreover, the temperature-dependent EPS production and originally conceived ability of flavins to act as electron shuttles suggest that not much complexity in bacteria with minerals. Additionally, the electrochemical potential was severely affected upon removal of EPS/flavin moiety from the intact biofilm, revealed the necessity of EPS bound flavins in transferring the electrons across its thick cell walls. This paradigm shift to electrogenic nature of Geobacillus sp. biofilm will become evident in the adaptation of other microbes during mineral respiration in extreme environments.

Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs.

We explore the possibility of characterizing sperm cells without the need to stain them using spectral and fluorescence lifetime analyses after multi-photon excitation in an insect model. The autofluorescence emission spectrum of sperm of the common bedbug, Cimex lectularius, was consistent with the presence of flavins and NAD(P)H. The mean fluorescence lifetimes showed smaller variation in sperm extracted from the male (tau m, τm = 1.54–1.84 ns) than in that extracted from the female sperm storage organ (tau m, τm = 1.26–2.00 ns). The fluorescence lifetime histograms revealed four peaks. These peaks (0.18, 0.92, 2.50 and 3.80 ns) suggest the presence of NAD(P)H and flavins and show that sperm metabolism can be characterized using fluorescence lifetime imaging. The difference in fluorescence lifetime variation between the sexes is consistent with the notion that female animals alter the metabolism of sperm cells during storage. It is not consistent, however, with the idea that sperm metabolism represents a sexually selected character that provides females with information about the male genotype.

Controlling electron transfer at the microbe–mineral interface

Remarkably, many bacteria live and thrive in the earth’s subsurface by respiring extracellular insoluble minerals. Okamato et al. (1) in PNAS report how this process may be accelerated by the presence of flavins that bind as cofactors to electron transport proteins on the cell surface that are key to extracellular mineral respiration.

Flavin-induced photodecomposition of sulfur-containing amino acids is decisive in the formation of beer lightstruck flavor

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.

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture.

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain of Pseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F420, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F420 was three times that of FAD. Efficient utilization of alcohols in the presence of F420 is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between the Pseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance

Visible light-induced photooxidation of glucose sensitized by riboflavin

We conducted this study to evaluate the oxidation of glucose induced by visible light in the presence of sensitizers such as methylene blue and flavins (i.e., flavin mononucleotide and riboflavin). The concentration of the sensitizers was similar to that of flavin in parenteral nutrients. The photooxidation of glucose sensitized by flavin mononucleotide or riboflavin was greater than that which was observed in the presence of methylene blue, whereas the isotopic effect of deuterium oxide (D 2O) was enhanced more substantially in the presence of methylene blue than in the presence of flavins. These results show that methylene blue exerts its action through singlet oxygen and that at a high substrate concentration (as was used in this work) flavin mononucleotide and riboflavin act preferentially as type I sensitizers. In the flavin photosensitized processes, the presence of hydrogen peroxide, superoxide anion, and hydroxyl radical was demonstrated. The photooxidation of glucose is favored by an increase in pH, and it also depends on the energy absorbed by the system. By using a specific reagent for glucose (i.e., o-toluidine), it was possible to quantify the photoconversion of glucose. The results obtained in this work should be considered in the management of glucose-containing parenteral nutrients that are exposed to visible light in the presence of a multivitamin complex containing flavin mononucleotide.

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors.

The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases.

Solubilization and ion-exchange chromatography of plasma membrane proteins obtained from bean (Phaseolus vulgaris L.) seedlings resulted in a single NAD(P)H-O2--synthase protein peak. This enzyme showed a high preference toward NADPH as a substrate (reaction rate, 27.4 nmol O2- produced min-1 mg-1 protein), whereas NADH reactions ranged from 0 to maximally 15% of the NADPH reactions. The protein functions as an oxidase and it was clearly resolved from NAD(P)H dehydrogenases identified with commonly used strong oxidants (ferricyanide, cytochrome c, DCIP, and oxaloacetate). The involvement of peroxidases in O2- production is excluded on the basis of potassium-cyanide insensitivity and NADPH specificity. The NADPH oxidase is only moderately stimulated by flavins (1.5-fold with 25 [mu]M flavine adenine dinucleotide and 2.5-fold with 25 [mu]M flavin mononucleotide) and inhibited by 100 [mu]M p-chloromercuribenzenesulfonic acid, 200 [mu]M diphenyleneiodonium, 10 mM quinacrine, 40 mM pyridine, and 20 mM imidazole. The presence of flavins was demonstrated in the O2-synthase fraction, but no b-type cytochromes were detected. The effect of these inhibitors and the detection of flavins and cytochromes in the plant O2- synthase make it possible to compare this enzyme with the NADPH O2- synthase of animal neutrophil cells.

Catalytic Action of Azolium Salts. VIII. Oxidative Aroylation with Arenecarbaldehydes Catalyzed by 1,3-Dimethylbenzimidazolium Iodide.

Refluxing of a mixture of benzaldehyde (1a), 1, 3-dimethylbenzimidazolium iodide (7), p-nitroaniline (9b) as an oxidizing agent, and 1, 8-diazabicyclo[5, 4, 0]-7-undecene (DBU) in MeOH afforded methyl benzoate (2a) in good yield. Other methyl arenecarboxylates 2 were similarly obtained from arenecarbaldehydes 1. We showed that this aroylation proceeds via the 2-aroyl-1, 3-dimethylbenzimidazolium salt (8). The 1, 2, 4-triazolium salt (18) and the naphtho[1, 2-d]imidazolium salt (19) were also effective catalysts for this oxidative aroylation. However, the aroylation did not proceed with the imidazolium salt (20). In the presence of flavins (25a-c), arenecarbaldehydes 1 reacted in MeOH under aerobic conditions catalyzed by the benzimidazolium salt 7 to give the corresponding methyl arenecarboxylates 2. 1-Methyl-3-[3-(10-phenylisoalloxazin-3-yl)propyl]benzimidazolium bromide (27) is an effective complex catalyst for this oxidative aroylation.

Partial purification of a plasma membrane flavoprotein and NAD(P)H‐oxidoreductase activity

Plasma membrane flavins and pterins are considered to mediate important physiological functions such as blue light photoperception and redox activity. Therefore, the presence of flavins and pterins in the plasma membrane of higher plants was studied together with NAD(P)H‐dependent redox activities. Plasma membranes were isolated from the apical hooks of etiolated bean seedlings (Phaseolus vulgaris L. cv. Limburgse Vroege) by aqueous two‐phase partitioning. Fluorescence spectroscopy revealed the presence of two chromophores. The first showed excitation maxima at 370 and 460 nm and an emission peak at 520 nm and was identified as a flavin. The second chromophore was probably a pterin molecule with excitation peaks at 290 and 350 nm and emission at 440 nm. Both pigments are considered intrinsic to the plasma membrane since they could not be removed by treatment with hypotonic media containing high salt and low detergent concentrations. The flavin concentration was estimated at about 500 pmol mg−1 protein. However difficulties were encountered in quantifying the pterin concentrations. Protease treatments indicated that the flavins were non‐covalently bound to the proteins. Separation of the plasma membrane proteins after solubilisation by octylglucoside, on an ion exchange system (HPLC, Mono Q), resulted in a distinct protein fraction showing flavin and pterin fluorescence and NADH oxidoreductase activity. The flavin of this fraction was identified as flavin mononucleotide (FMN) by HPLC analysis. Other minor peaks of NADH:acceptor reductase activity were resolved on the column. The presence of distinct NAD(P)H oxidases at the plasma membrane was supported by nucleotide specificity and latency studies using intact vesicles. Our work demonstrates the presence of plasma membrane flavins as intrinsic chromophores, that may function in NAD(P)H‐oxidoreductase activity and suggests the presence of plasma membrane bound pterins.

Covalent Attachment of FAD Derivatives to a Fusion Protein Consisting of 6-Hydroxy-D-nicotine Oxidase and a Mitochondrial Presequence: FOLDING, ENZYME ACTIVITY, AND IMPORT OF THE MODIFIED PROTEIN INTO YEAST MITOCHONDRIA

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N6-(2-aminoethyl)-FAD and N6-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F0-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD. The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

Flavin-sensitized photooxidation of substituted phenols in natural water

Photooxidation of substituted phenols in the presence of flavins as sensitizers was investigated under aerobic conditions to determine the fate of synthetic chemicals in the environment. Riboflavin was easily decomposed: lumichrome formed after several minutes of illumination with simulated sunlight. In contrast, lumichrome was extremely stable toward sunlight, and was found to be a major flavin component in natural water. From the result of the lumichrome-sensitized photodecomposition of substituted phenols, the order of photolysis rate is p- methoxyphenol > p- chlorophenol > phenol > p- nitrophenol . A decrease in total organic carbon (TOC) from the reaction solutions of all phenols except for p- nitrophenol was observed, and the carbon released was found to mainly originate from phenols. The compound p- chlorophenol has been shown to degrade readily by lumichrome-sensitized photooxidation, but the transformation occurs only by dechlorination. Polychlorophenols were completely dechlorinated in ∼ 1 hr after lumichrome-sensitized photolysis, and the rate increased with chlorine content on the aromatic ring.

On the reaction mechanism of flavin-sensitized photoregulation of Monoraphidium braunii nitrate reductase

Nitrate reductase from the green alga Monoraphidium braunii is reversibly inactivated by reduction on irradiation in the presence of flavins and an electron donor; the process is markedly accelerated by cyanide anions. When the enzyme is depleted of its endogenous flavin adenine dinucleotide (FAD), a significant lag period of 1–2 min is observed before photoinactivation takes place. This lag period is not observed if flavins (except FAD) are previously photoreduced in the reaction mixture; in this case the inactivation kinetics depend on the type of flavin used. The photoinactivation process is accompanied by reduction of the haem prosthetic groups. The flavin-sensitized photoinactivation of nitrate reductase is dependent on ionic strength; this appears to be due to complementary electrostatic interactions between the reduction site of the protein and the exogenous flavins. Flavins can also photosensitize the reactivation of the cyanide-inactivated reduced enzyme (either native or FAD-depleted nitrate reductase) in the absence of added electron donors. The mechanism of flavin-mediated nitrate reductase photoregulation is discussed.

FLAVINS AND QUINONES AS ELECTRON TRANSFER MEDIATORS FOR DIAPHORASE CATALYZED ELECTROLYTIC OXIDATION OF NADH

Diaphorase (DI)-catalyzed electrolytic oxidation of NADH to NAD + is studied in the presence of flavins and quinones as electron transfer mediators. The behavior of the catalytic current at the mediate DI-electrode is well explained by theoretical equations based on polarographic catalytic current theory using the reaction layer approximation. The bimolecular reaction rate constants between DI and the mediators are determined by the electrochemical method

Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography

The presence of flavins in the retina has been known for some time. However, the small size of the tissue has made it difficult to quantify the levels of the individual flavins, riboflavin (RB), FMN, and FAD without pooling large numbers of retinas. A procedure to extract and quantitate RB, FMN, and FAD in retinal tissue from as few as four rat retinas has been developed. The procedure resolves these three classes of flavins and provides a recovery near 100%. For the analysis, HPLC using a reverse-phase column with cyclohexyl functional groups was coupled to a fluorescence detector. The microextraction-HPLC procedure was reproducible for the quantitative analysis of flavins in the retina and equally applicable for analysis of flavins in liver and plasma.

Flavin-mediated photolysis of mycosporines

Under aerobic conditions and in the presence of flavins, light causes the photolysis of mycosporine glutamine into aminocyclohexenone and 2-hydroxy glutaric acid. This photolysis, which is temperature dependent, is also observed when other photosensitizers which are carriers of singlet oxygen replace flavins. This photodestruction also occurs with mycosporine amino alcohols but at reduced rate and probably by another mechanism.

Photooxidation of hypotaurine to taurine in the presence of flavins.

Irradiation of organic sulfinates such as hypotaurine and cysteine sulfinic acid in the presence of catalytic amounts of flavins led to the oxidation of its sulfinic groups (-SO2H) to the corresponding sulfonates. The process of hypotaurine oxidation in the presence of riboflavin, followed by absorbance decrease at 220 nm and by ion-exchange chromatography, showed a pseudo-first-order kinetics at pH 6.0. The k value depended linearly on flavin concentrations. The reaction rate was higher at acidic pH. Although the reaction rate was not affected by the addition of superoxide dismutase or catalase, superoxide ions were supposed to be by-products of the reaction. The effectiveness of allyl alcohol as a scavenger pointed to a free-radical mechanism of the reaction. We propose a new reaction mechanism involving sulfinic radicals on this photochemical reaction.