Free Flavin Adenine Dinucleotide Research Articles

Flavinated SDHA underlies the change in intrinsic optical properties of oral cancers

The molecular basis of reduced autofluorescence in oral squamous cell carcinoma (OSCC) cells relative to normal cells has been speculated to be due to lower levels of free flavin adenine dinucleotide (FAD). This speculation, along with differences in the intrinsic optical properties of extracellular collagen, lies at the foundation of the design of currently-used clinical optical detection devices. Here, we report that free FAD levels may not account for differences in autofluorescence of OSCC cells, but that the differences relate to FAD as a co-factor for flavination. Autofluorescence from a 70 kDa flavoprotein, succinate dehydrogenase A (SDHA), was found to be responsible for changes in optical properties within the FAD spectral region, with lower levels of flavinated SDHA in OSCC cells. Since flavinated SDHA is required for functional complexation with succinate dehydrogenase B (SDHB), decreased SDHB levels were observed in human OSCC tissue relative to normal tissues. Accordingly, the metabolism of OSCC cells was found to be significantly altered relative to normal cells, revealing vulnerabilities for both diagnosis and targeted therapy. Optimizing non-invasive tools based on optical and metabolic signatures of cancers will enable more precise and early diagnosis leading to improved outcomes in patients.

Electrochemical Response of Glucose Oxidase Adsorbed on Laser-Induced Graphene.

Carbon-based electrodes have demonstrated great promise as electrochemical transducers in the development of biosensors. More recently, laser-induced graphene (LIG), a graphene derivative, appears as a great candidate due to its superior electron transfer characteristics, high surface area and simplicity in its synthesis. The continuous interest in the development of cost-effective, more stable and reliable biosensors for glucose detection make them the most studied and explored within the academic and industry community. In this work, the electrochemistry of glucose oxidase (GOx) adsorbed on LIG electrodes is studied in detail. In addition to the well-known electroactivity of free flavin adenine dinucleotide (FAD), the cofactor of GOx, at the expected half-wave potential of −0.490 V vs. Ag/AgCl (1 M KCl), a new well-defined redox pair at 0.155 V is observed and shown to be related to LIG/GOx interaction. A systematic study was undertaken in order to understand the origin of this activity, including scan rate and pH dependence, along with glucose detection tests. Two protons and two electrons are involved in this reaction, which is shown to be sensitive to the concentration of glucose, restraining its origin to the electron transfer from FAD in the active site of GOx to the electrode via direct or mediated by quinone derivatives acting as mediators.

Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH, FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques.

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.

Intracellular free flavin and its associated enzymes participate in oxygen and iron metabolism in Amphibacillus xylanus lacking a respiratory chain

Amphibacillus xylanus is a recently identified bacterium which grows well under both aerobic and anaerobic conditions and may prove useful for biomass utilization. Amphibacillus xylanus, despite lacking a respiratory chain, consumes oxygen at a similar rate to Escherichia coli (130–140 μmol oxygen·min−1·g−1 dry cells at 37 °C), suggesting that it has an alternative system that uses a large amount of oxygen. Amphibacillus xylanus NADH oxidase (Nox) was previously reported to rapidly reduce molecular oxygen content in the presence of exogenously added free flavin. Here, we established a quantitative method for determining the intracellular concentrations of free flavins in A. xylanus, involving French pressure and ultrafiltration membranes. The intracellular concentrations of flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and riboflavin were estimated to be approximately 8, 3, and 1 μm, respectively. In the presence of FAD, the predominant free flavin species, two flavoproteins Nox (which binds FAD) and NAD(P)H oxidoreductase (Npo, which binds FMN), were identified as central free flavin‐associated enzymes in the oxygen metabolic pathway. Under 8 μm free FAD, the catalytic efficiency (k cat/K m) of recombinant Nox and Npo for oxygen increased by approximately fivefold and ninefold, respectively. Nox and Npo levels were increased, and intracellular FAD formation was stimulated following exposure of A. xylanus to oxygen. This suggests that these two enzymes and free FAD contribute to effective oxygen detoxification and NAD(P)+ regeneration to maintain redox balance during aerobic growth. Furthermore, A. xylanus required iron to grow aerobically. We also discuss the contribution of the free flavin‐associated system to the process of iron utilization.

Characterization of wheat endoplasmic reticulum oxidoreductin 1 and its application in Chinese steamed bread

This study investigated characteristics of recombinant wheat Endoplasmic Reticulum Oxidoreductin 1 (wEro1) and its influence on Chinese steamed bread (CSB) qualities. The purified wEro1 monomer, which contained two conserved redox active motif sites, bound to flavin adenine dinucleotide (FAD) cofactor with a molecular weight of ∼47 kDa. wEro1 catalyzed the reduction of both bound and free FAD, and its reduction activity of free FAD reached 7.8 U/mg. Moreover, wEro1 catalyzed the oxidation of dithiothreitol and wheat protein disulfide isomerase (wPDI). Both glutathione and the reduced ribonuclease could work as electron donors for wEro1 in catalyzing the oxidation of wPDI. Additionally, wEro1 supplementation improved the CSB qualities with an increased specific volume of CSB and decreased crumb hardness, which was attributed to water-insoluble wheat proteins increasing and gluten network strengthening. The results give an understanding of the properties and function of wEro1 to facilitate its application especially in the flour-processing industry.

Quantification of the Metabolic State in Cell-Model of Parkinson's Disease by Fluorescence Lifetime Imaging Microscopy.

Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson’s disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD, and their relative contributions in MPP+ (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP+ treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP+ treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP+ treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level.

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.

Effects of riboflavin supplementation on growth performance, carcass traits, and riboflavin status of growing male white Pekin ducks

An experiment involving six dietary riboflavin concentration (1.38, 2.38, 3.38, 4.38, 5.38, and 6.38mg/kg) was conducted to determine the effects of riboflavin on performance and riboflavin status of growing male white Pekin ducks in terms of growth performance, carcass traits, and free riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) concentration in the liver and plasma. A basal corn-soybean meal containing 1.38mg/kg of naturally occurring riboflavin was used and this basal diet was fed with 0, 1, 2, 3, 4, and 5mg/kg diet of supplemental riboflavin. One-day-old male white Pekin ducks were fed common starter diets from hatch to 14 days of age and then fed the experimental diets from 15 to 35 days of age. A total of 288 15-day-old ducks were allotted to 6 dietary treatments with 6 replicate pens of 8 birds per pen. At 35 days of age, the average daily weight gain (ADG), average daily feed intake (ADFI), gain to feed ratio (G:F), and percentage yield of breast and leg meat, abdominal fat were examined and free riboflavin, FMN, and FAD in plasma or liver were all analyzed. The growth depression, low plasma free riboflavin, liver free riboflavin, liver FMN, and liver FAD concentration were observed in the ducks fed the riboflavin-deficient basal diet (P<0.05), and these adverse effects were alleviated by riboflavin supplementation. The growing ducks fed the basal diet with no supplementation of riboflavin had the lowest ADG, ADFI, G:F and breast meat yield among all ducks (P<0.05) and these criteria showed linear or quadratic response to increasing dietary riboflavin (P<0.05). On the other hand, dietary riboflavin influenced the status of this vitamin in growing ducks. The poorest growth performance of the riboflavin-deficient growing ducks was accompanied with the lowest content of riboflavin, FAD, and FMN in plasma or liver (P<0.05) but these bad statuses were reversed by increasing dietary riboflavin in which the riboflavin and its derivative increased linearly or quadratically as dietary riboflavin increased (P<0.05). According to the broken-line model, the riboflavin requirements (based on dietary total riboflavin) of male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, G:F, plasma free riboflavin, liver free riboflavin, and liver FMN were 2.43, 2.48, 2.31, 3.57, 2.78, and 2.83mg/kg, respectively.

Fluorescence lifetime imaging ophthalmoscopy in type 2 diabetic patients who have no signs of diabetic retinopathy.

The time-resolved autofluorescence of the eye is used for the detection of metabolic alteration in diabetic patients who have no signs of diabetic retinopathy. One eye from 37 phakic and 11 pseudophakic patients with type 2 diabetes, and one eye from 25 phakic and 23 pseudophakic healthy subjects were included n the study. After a three-exponential fit of the decay of autofluorescence, histograms of lifetimes τ(i), amplitudes α(i), and relative contributions Q(i) were statistically compared between corresponding groups in two spectral channels (490 < ch1 < 560 nm, 560 < ch2 < 700 nm). The change in single fluorophores was estimated by applying the Holm–Bonferroni method and by calculating differences in the sum histograms of lifetimes. Median and mean of the histograms of τ(2), τ(3), and α(3) in ch1 show the greatest differences between phakic diabetic patients and age-matched controls (p < 0.000004). The lack of pixels with a τ(2) of ∼360 ps, the increased number of pixels with τ(2) > 450 ps, and the shift of τ(3) from ∼3000 to 3700 ps in ch1 of diabetic patients when compared with healthy subjects indicate an increased production of free flavin adenine dinucleotide, accumulation of advanced glycation end products (AGE), and, probably, a change from free to protein-bound reduced nicotinamide adenine inucleotide at the fundus. AGE also accumulated in the crystalline lens.

Corneal Metabolic State Assessment by Fluorescence Lifetime Imaging Microscopy

A long time objective of ophthalmologists is to diagnose corneal cells dysfunction prior to its pathologic expression. With this motivation, we are currently developing a new instrument for in vivo metabolic imaging of corneal tissues.Metabolic alterations are known to be the first sign of several corneal pathologies and can be assessed through non-invasive monitoring of metabolic co-factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). The quantification of the relative proportions between free and protein-bound NADH and FAD can be achieved using fluorescence lifetime-resolved methods. This approach has already been applied in age-related macular degeneration, diabetic retinopathy and epithelial cancer.FAD and NADH imaging can be performed by one-photon excitation (1PE) and two-photon excitation (2PE) techniques. The latest has the advantage of allowing simultaneous excitation of both metabolic co-factors. However, there are still safety concerns when considering in vivo ocular studies in humans using 2PE.Due to these concerns we used, as a first approach, a 1PE system for evaluating the feasibility of corneal FAD imaging. The use of FAD has advantages over NADH. It can be excited over longer excitation wavelengths, is more resistant to photo-bleaching and is located exclusively in the mitochondrial space.A PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan) was used to monitor FAD autofluorescence. The instrument uses a 440 nm pulsed diode laser (330 ps) running at a pulse rate of 40 MHz. The instrument was modified by us to allow the acquisition of both fluorescence lifetime and reflectance images and to enhance scattered light rejection.Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH, Berlin, Germany). The fluorescence decay times were obtained after applying a non-linear least square fit to the decay data and the goodness of fit was evaluated by the analysis of the residuals and the chi-squared (χ2).We have acquired fluorescence lifetime images of ex vivo healthy Wistar rat corneas (Fig.1) using two different instrument setups: 1- using the emission filters provided by the manufacturer; 2- placing extra emission filters to fully reject the scattered excitation light. In both setups, FAD fluorescence data presented a bi-exponential decay with a short (protein-bound FAD) and a longer (free FAD) lifetime component.While both setups provide FAD fluorescence decays, only the second retrieves valid metabolic information. We obtained two lifetime components, one of 0.118 (0.028) ns and another of 2.11 (0.16) ns, with a relative contributions of 39.4 (2.2) and 60.6 (2.2), respectively. These values are in accordance with the literature.Corneal layer discrimination is possible based on morphologic characteristics. However, the fluorescence lifetime images do not provide morphological detail (Fig.1), possibly because FAD is only present in the mitochondria. These organelles are small and tend to accumulate around the nuclei.So, we modified the instrument’s optical setup to allow the acquisition of both fluorescence lifetime images and reflectance images. Figure 2 shows an example of the corneal epithelial layer.The image resolution and depth penetration are still not ideal. Since the assessment of corneal endothelial layer metabolic function is also within our goals, we are currently implementing further modifications to improve both the instrument’s resolution and depth penetration.The characterization of FAD fluorescence lifetime in unhealthy corneas is important to detect corneal dysfunctions prior to its pathologic expression. Therefore, we intend to study metabolic altered Wistar rat corneas. The alterations will be induced by potassium cyanide, which is a reversible inhibitor of the fourth complex of the mitochondrial electron transport chain.Financial support received from the Fundação para a Ciência e a Tecnologia under the research projects PTDC/SAU-BEB/104183/2008 and PTDC/SAU-ENB/122128/2010.

Sensitive d-amino acid biosensor based on oxidase/peroxidase system mediated by pentacyanoferrate-bound polymer

A sensitive d-amino acid oxidase (DAAO)/peroxidase (POD) bienzyme biosensor is constructed, in which pentacyanoferrate-bound poly(1-vinylimidazole) polymer (PVI[Fe(CN)5]) is selected as a mediator. Reductive current of PVI[Fe(CN)5] related to the H2O2 concentration generated in the DAAO reaction was measured at −0.1V vs. Ag|AgCl with DAAO/POD/PVI[Fe(CN)5]-modified electrode. The result revealed that PVI[Fe(CN)5] is suitable as a mediator for this bienzyme system due to its appropriate formal potential and its extremely low reactivity against DAAO. The stability of DAAO was improved by adding free flavin adenine dinucleotide and the electrode composition was optimized for the detection of d-alanine. Nafion and ascorbate oxidase-immobilized films worked successfully to prevent severe interference from uric acid and ascorbic acid. The low detection limits of d-alanine (2μM) and d-serine (2μM) imply its possibility for the determination of extremely low concentration of d-amino acids in physiological fluids. The proposed bienzyme biosensor is proved to be capable of detecting d-amino acids in urine.

A hybrid FLIM-elastic net platform for label free profiling of breast cancer

We report a label-free fluorescence lifetime profiling strategy to classify breast cancer cells, MCF10CA1h (malignant), MCF10A (nonmalignant), and MCF10AneoT (premalignant) in different stages of malignancy. Fluorescence Lifetime Imaging Microscopy (FLIM) was used to record the lifetime of autofluorescence of endogenous flavin in MCF10 cells in different stages of malignancy. Predominant differences in lifetimes ascertained by multi-exponential fitting curves can be attributed to the different forms of flavin protein; flavin mononucleotide (FMN), free flavin adenine dinucleotide (FAD), semiquinone, and bound FAD. A lifetime map of the metabolite was derived from the contribution of the lifetime of each metabolite by iterative reconvolution fitting of the Time Correlated Single Photon Counting (TCSPC) decay curves. Lifetime maps were constructed by mapping the average lifetime values pixel by pixel using MATLAB. The FLIM image (150 × 150 pixels) of each cell was extracted, resized and centered into 100 × 100 pixels using the nearest neighbor algorithm. Principal Component Analysis (PCA) in conjunction with Elastic net Analysis (EnA) was then used to classify the different stages of MCF10 cell lines based on average lifetime values. The EnA model provided an excellent classification of the cells at different stages of tumorigenesis yielding 100% accuracy.

Corneal respiratory function by FAD autofluorescence lifetime

AbstractPurpose We intend to develop an efficient method to assess in‐vivo the corneal respiratory function in order to diagnose corneal cells dysfunction prior to its pathologic expression.Methods Metabolic alterations can be assessed by measuring the amount of the metabolic co‐factors flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH). FAD has advantages over NADH, like being present only in the mitochondria. Furthermore, using fluorescence lifetime imaging microscopy (FLIM) we are able to discriminate between its free or protein‐bound states. We resorted to a PicoQuant MicroTime 100 (PicoQuant GmbH, Berlin, Germany) coupled to an Olympus BX51 Microscope (Olympus Corporation, Tokyo, Japan). This setup uses a pulsed blue diode laser with a trigger frequency of 40 MHz with an excitation filter of 440±10 nm. Intensity decay curves were processed with SymPhoTime v5.3 Software (PicoQuant GmbH). The instrument response function was acquired to improve data analysis precision.Results We successfully acquired ex‐vivo autofluorescence images of male Wistar rat and Bovine corneas. A bi‐exponential decay was observed in both cases with a fast decay around 1 ns and a longer one around 4 ns, which correspond to protein‐bound and free FAD, respectively. These results are in accordance with other studies, although there is some controversy regarding FAD lifetimes.Conclusion We showed that it is possible, with our apparatus, to acquire metabolic images of the cornea using FAD autofluorescence. We intend to modify the instrument optical setup in order to acquire reflectance and fluorescence lifetime images simultaneously for corneal layer identification.

Ectopic Expression of Riboflavin-binding Protein Gene TsRfBP Paradoxically Enhances Both Plant Growth and Drought Tolerance in Transgenic Arabidopsis thaliana

Riboflavin is the precursor of the coenzymes flavin monophosphate (FMN) and flavin adenine dinucleotide (FAD), which serve as indispensable redox cofactors in all plants. Numerous data indicate that riboflavin is involved in pathogen resistance but less data are available on abiotic stress tolerance. In this experiment, the overexpression of the riboflavin-binding protein resulted in an enhancement of vegetative growth and net photosynthetic rate, and an acceleration of floral transition in transgenic Arabidopsis thaliana REAT11 (containing less than half the normal levels of free riboflavin, FMN, and FAD) compared to wild-type Col-0 under nonstressed conditions. The effect of drought stress on the antioxidant response of Col-0 and REAT11 was compared, where 20- and 40-day-old grown plants were subjected to 10 % PEG 6000 treatment for 2 days. Stress conditions caused a significant increase in H2O2 accumulation, lipid peroxidation, and membrane permeability in Col-0 over that in REAT11. Greater activity levels of superoxide dismutase, ascorbate peroxidase, and glutathione reductase were observed in the leaves of REAT11 compared to those of Col-0. Significant increases in total ascorbate and glutathione content and higher ratios of ASC/DHA: (ASC and DHA are reduced and oxidized ascorbate, respectively) and GSH/GSSG: (GSH and GSSG are reduced and oxidized glutathione, respectively) were observed in the leaves of REAT11 compared to those in Col-0 under drought conditions. In addition, enhancement of free proline and soluble sugar accumulation was observed in REAT11 compared to Col-0 under stress. Our results suggest that a slight deficiency in free riboflavin can paradoxically induce both a higher vegetative growth rate and an enhanced tolerance to drought in transgenic plants. The “stress escape” hypothesis is proposed here to explain this interesting phenomenon.

Properties and Crystal Structure of Methylenetetrahydrofolate Reductase from Thermus thermophilus HB8

BackgroundMethylenetetrahydrofolate reductase (MTHFR) is one of the enzymes involved in homocysteine metabolism. Despite considerable genetic and clinical attention, the reaction mechanism and regulation of this enzyme are not fully understood because of difficult production and poor stability. While recombinant enzymes from thermophilic organisms are often stable and easy to prepare, properties of thermostable MTHFRs have not yet been reported.Methodology/Principal FindingsMTHFR from Thermus thermophilus HB8, a homologue of Escherichia coli MetF, has been expressed in E. coli and purified. The purified MTHFR was chiefly obtained as a heterodimer of apo- and holo-subunits, that is, one flavin adenine dinucleotide (FAD) prosthetic group bound per dimer. The crystal structure of the holo-subunit was quite similar to the β8α8 barrel of E. coli MTHFR, while that of the apo-subunit was a previously unobserved closed form. In addition, the intersubunit interface of the dimer in the crystals was different from any of the subunit interfaces of the tetramer of E. coli MTHFR. Free FAD could be incorporated into the apo-subunit of the purified Thermus enzyme after purification, forming a homodimer of holo-subunits. Comparison of the crystal structures of the heterodimer and the homodimer revealed different intersubunit interfaces, indicating a large conformational change upon FAD binding. Most of the biochemical properties of the heterodimer and the homodimer were the same, except that the homodimer showed ≈50% activity per FAD-bound subunit in folate-dependent reactions.Conclusions/SignificanceThe different intersubunit interfaces and rearrangement of subunits of Thermus MTHFR may be related to human enzyme properties, such as the allosteric regulation by S-adenosylmethionine and the enhanced instability of the Ala222Val mutant upon loss of FAD. Whereas E. coli MTHFR was the only structural model for human MTHFR to date, our findings suggest that Thermus MTHFR will be another useful model for this important enzyme.

Factor analysis of spectroelectrochemical reduction of FAD reveals the pKa of the reduced state and the reduction pathway

AbstractThe free flavin adenine dinucleotide (FAD) cofactor is known to exhibit a pH‐dependent midpoint potential involving a simultaneous two‐electron transfer step (n = 2). Uv‐vis spectroelectrochemical reductions of FAD at constant pH, ranging from 5 to 9, were recorded and analyzed by factor analysis. Principal factor analysis was used to determine the number of species present at each pH. The results indicate that only two composite forms of FAD are present: the oxidized and the reduced forms. Window factor analysis was used to extract the concentration profiles of the controlling species. The oxidized form was found to be a single pH‐independent species, whereas the reduced form consists of two species. The pH‐dependent spectroscopic changes of reduced FAD were best modeled by a single proton transfer step involving two different ionization states with an apparent pKa = 6.3. This value compares favorably with those obtained from NMR and from midpoint potential measurements. At pH 6, the reduction of FAD was found to be first order, whereas at pH 9 the reduction is zero order; these observations are explained in terms of the reaction pathway involving xanthine oxidase, its substrate, and the pH. Copyright © 2007 John Wiley &amp; Sons, Ltd.

Study on Direct Electrochemistry of Glucose Oxidase Stabilized by Cross‐Linking and Immobilized in Silica Nanoparticle Films

AbstractGlucose oxidase (GOD) in SiO2 nanoparticle films was cross‐linked by glutaraldehyde (GA), forming GOD‐GA‐SiO2 films on the surface of pyrolytic graphite (PG) electrodes. GOD in the GOD‐GA‐SiO2 films showed a pair of well defined and quasi‐reversible cyclic voltammetric peaks at −0.48 V vs. SCE in pH 7.0 buffers. Scanning electron microscopy of the films demonstrated that the interaction between GOD and GA/SiO2 would make the morphology of dry GOD‐GA‐SiO2 films very different from that of SiO2 and GOD‐SiO2 films. IR spectroscopy confirms that the GODs in the films are indeed cross‐linked through GA, and GA in the films greatly improves the film stability. UV‐vis and IR spectroscopy suggest that the enzyme is not denatured by cross‐linking in the films. The comparative bioelectrocatalytic study with free flavin adenine dinucleotide (FAD) in SiO2 films indicates that GOD in the films retains its biocatalytic activity toward glucose oxidation. All these results show that SiO2 nanoparticle films not only provide a favorable microenvironment for the native GOD to exchange electrons directly with underlying electrodes, but also supply a matrix for stabilizing GOD when combined with GA.

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

Spectrally resolved time-correlated single photon counting: a novel approach for characterization of endogenous fluorescence in isolated cardiac myocytes

A new setup for time-resolved fluorescence micro-spectroscopy of cells, based on multi-dimensional time-correlated single photon counting, was designed and tested. Here we demonstrate that the spectrometer allows fast and reproducible measurements of endogenous flavin fluorescence measured directly in living cardiac cells after excitation with visible picosecond laser diodes. Two complementary approaches for the analysis of spectrally- and time-resolved autofluorescence data are presented, comprising the fluorescence decay fitting by exponential series and the time-resolved emission spectroscopy analysis. In isolated cardiac myocytes, we observed three distinct lifetime pools with characteristic lifetime values spanning from picosecond to nanosecond range and the time-dependent red shift of the autofluorescence emission spectra. We compared obtained results to in vitro recordings of free flavin adenine dinucleotide (FAD) and FAD in lipoamide dehydrogenase (LipDH). The developed setup combines the strength of both spectral and fluorescence lifetime analysis and provides a solid base for the study of complex systems with intrinsic fluorescence, such as identification of the individual flavinoprotein components in living cardiac cells. This approach therefore constitutes an important instrumental advancement towards redox fluorimetry of living cardiomyocytes, with the perspective of its applications in the investigation of oxidative metabolic state under pathophysiological conditions, such as ischemia and/or metabolic disorders.

Light-Induced Structural Changes of Apoprotein and Chromophore in the Sensor of Blue Light Using FAD (BLUF) Domain of AppA for a Signaling State

AppA is a new class blue-light receptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides and retains a characteristic flavin adenine dinucleotide (FAD)-binding domain named the "sensor of blue light using FAD" (BLUF). AppA functions as an antirepressor controlling transcription of photosynthesis genes through the direct association with a transcriptional repressor PpsR in a blue-light-dependent manner [Masuda and Bauer (2002) Cell 110, 613-623]. Illumination of AppA induces a red shift in the UV-visible absorption of FAD, which results in a signaling state of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUF domain showed relatively simple features, which were mainly composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm(-)(1). We have developed an in vitro reconstitution method, by which a fully functional BLUF domain was reconstituted from free FAD and an apoprotein for the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopically labeled with (13)C and unlabeled FAD was constituted using this method, and hydrated and deuterated samples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domain were compared with those for uniformly (15)N- and (13)C-labeled or deuterated domains as well as for the unlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore and apoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes were discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.