flavin-adenine-dinucleotide and flavin-semiquinone

Flavin semiquinone redox states are important intermediates in a broad variety of reactions catalyzed by flavoproteins. As paramagnetic states they can be favorably probed by EPR spectroscopy in all its flavors. This review summarizes recent results in the characterization of flavin radicals. On the one hand, flavin radical states, e.g., trapped as reaction intermediates, can be characterized using modern pulsed EPR methods to unravel their electronic structure and to gain information about the surrounding environment and its changes on protein action. On the other hand, short-lived intermediate flavin radical states generated, e.g., photochemically, can be followed by time-resolved EPR, which allows a direct tracking of flavin-dependent reactions with a temporal resolution reaching nanoseconds.

Topics: Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Flavoproteins; Oxidation-Reduction

Iodotyrosine deiodinase (IYD) is unusual for its reliance on flavin to promote reductive dehalogenation under aerobic conditions. As implied by the name, this enzyme was first discovered to catalyze iodide elimination from iodotyrosine for recycling iodide during synthesis of tetra- and triiodothyronine collectively known as thyroid hormone. However, IYD likely supports many more functions and has been shown to debrominate and dechlorinate bromo- and chlorotyrosines. A specificity for halotyrosines versus halophenols is well preserved from humans to bacteria. In all examples to date, the substrate zwitterion establishes polar contacts with both the protein and the isoalloxazine ring of flavin. Mechanistic data suggest dehalogenation is catalyzed by sequential one electron transfer steps from reduced flavin to substrate despite the initial expectations for a single two electron transfer mechanism. A purported flavin semiquinone intermediate is stabilized by hydrogen bonding between its N5 position and the side chain of a Thr. Mutation of this residue to Ala suppresses dehalogenation and enhances a nitroreductase activity that is reminiscent of other enzymes within the same structural superfamily.

Topics: Animals; Flavin-Adenine Dinucleotide; Humans; Iodide Peroxidase; Iodides; Nitroreductases; Triiodothyronine

Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented.

Topics: Amino Acid Sequence; Anions; Biocatalysis; Flavin-Adenine Dinucleotide; Mixed Function Oxygenases; Models, Molecular; Molecular Sequence Data; Oxidation-Reduction; Protein Conformation; Sequence Homology, Amino Acid; Substrate Specificity

Bifurcating electron transfer flavoproteins (Bf ETFs) are important redox enzymes that contain two flavin adenine dinucleotide (FAD) cofactors, with contrasting reactivities and complementary roles in electron bifurcation. However, for both the "electron transfer" (ET) and the "bifurcating" (Bf) FADs, the only charged amino acid within 5 Å of the flavin is a conserved arginine (Arg) residue. To understand how the two sites produce different reactivities utilizing the same residue, we investigated the consequences of replacing each of the Arg residues with lysine, glutamine, histidine, or alanine. We show that absence of a positive charge in the ET site diminishes accumulation of the anionic semiquinone (ASQ) that enables the ET flavin to act as a single electron carrier, due to depression of the oxidized versus. ASQ reduction midpoint potential, E°

Topics: Adenosine Monophosphate; Arginine; Electron Transport; Electron-Transferring Flavoproteins; Flavin-Adenine Dinucleotide; Flavins; Oxidation-Reduction

Electron transfer bifurcation allows production of a strongly reducing carrier at the expense of a weaker one, by redistributing energy among a pair of electrons. Thus, two weakly-reducing electrons from NADH are consumed to produce a strongly reducing ferredoxin or flavodoxin, paid for by reduction of an oxidizing acceptor. The prevailing mechanism calls for participation of a strongly reducing flavin semiquinone which has been difficult to observe with site-certainly in multi-flavin systems. Using blue light (450 nm) to photoexcite the flavins of bifurcating electron transfer flavoprotein (ETF), we demonstrate accumulation of anionic flavin semiquinone in excess of what is observed in equilibrium titrations, and establish its ability to reduce the low-potential electron acceptor benzyl viologen. This must occur at the bifurcating flavin because the midpoint potentials of the electron transfer (ET) flavin are not sufficiently negative. We show that bis-tris propane buffer is an effective electron donor to the flavin photoreduction, but that if the system is prepared with the ET flavin chemically reduced, so that only the bifurcating flavin is oxidized and photochemically active, flavin anionic semiquinone is formed more rapidly. Thus, excited bifurcating flavin is able to draw on an electron stored at the ET flavin. Flavin semiquinone photogenerated at the bifurcation site must therefore be accompanied by additional semiquinone formation by oxidation of the ET flavin. Consistent with the expected instability of bifurcating flavin semiquinone, it subsides immediately upon cessation of illumination. However comparison with yields of semiquinone in equilibrium titrations suggest that during continuous illumination at pH 9 a steady state population of 0.3 equivalents of bifurcating flavin semiquinone accumulates, and then undergoes further photoreduction to the hydroquinone. Although transient, the population of bifurcating flavin semiquinone explains the system's ability to conduct light-driven electron transfer from bis-tris propane to benzyl viologen, in effect trapping energy from light.

Topics: Bacterial Proteins; Electron Transport; Electron-Transferring Flavoproteins; Flavin-Adenine Dinucleotide; Flavins; Kinetics; Oxidation-Reduction; Photochemistry; Rhodopseudomonas

The electron configuration of flavin cofactors, FMN and FAD, is a critical factor governing the reactivity of NADPH-cytochrome P450 reductase (CPR). The current view of electron transfer by the mammalian CPR, based on equilibrium redox potentials of the flavin cofactors, is that the two electron-reduced FMN hydroquinone (FMNH2), rather than one electron-reduced FMN semiquinone, serves as electron donor to the terminal protein acceptors. However, kinetic and thermodynamic studies on the CPR species originated from different organisms have shown that redox potentials measured at distinct electron transfer steps differ from redox potentials determined by equilibrium titration. Collectively, previous observations suggest that the short-lived transient semiquinone species may carry electrons in diflavin reductases. In this work, we have investigated spectroscopic properties of the CPR-bound FAD and FMN reduced at 77 K by radiolytically-generated thermalized electrons. Using UV-vis spectroscopy, we demonstrated that upon cryo-reduction of oxidized yeast CPR (yCPR) containing an equimolar ratio of both FAD and FMN, or FAD alone, neutral semiquinones were trapped at 77 K. During annealing at the elevated temperatures, unstable short-lived neutral semiquinones relaxed to spectroscopically distinct air-stable neutral semiquinones. This transition was independent of pH within the 6.0-10.7 range. Our data on yeast CPR are in line with the previous observations of others that the flavin short-lived transient semiquinone intermediates may have a role in the electron transfer by CPR at physiological conditions.

Topics: Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Glucose Oxidase; Kinetics; NADPH-Ferrihemoprotein Reductase; Oxidation-Reduction; Temperature; Yeasts

Fluorescent cofactors like flavins can be exploited to probe their local environment with spatial and temporal resolution. Although the fluorescence properties of the oxidized and two-electron-reduced states of flavins have been studied extensively, this is not the case for the one-electron-reduced state. Both the neutral and anionic semiquinones have proven particularly challenging to examine, as they are unstable in solution and are transient, short-lived species in many catalytic cycles. Here, we report that the nitronate monooxygenase (NMO) from Pseudomonas aeruginosa PAO1 is capable of stabilizing both semiquinone forms anaerobically for hours, thus enabling us to study their spectroscopy in a constant protein environment. We found that in the active site of NMO, the anionic semiquinone exhibits no fluorescence, whereas the neutral semiquinone radical shows a relatively strong fluorescence, with a behavior that violates the Kasha-Vavilov rule. These fluorescence properties are discussed in the context of time-dependent density functional theory calculations, which reveal low-lying dark states in both systems.

Topics: Density Functional Theory; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Fluorescence; Free Radicals; Mixed Function Oxygenases; Models, Chemical; Oxidation-Reduction; Pseudomonas aeruginosa

There is an increasing realization that structure-based drug design may show improved success by understanding the ensemble of conformations accessible to an enzyme and how the environment affects this ensemble. Human monoamine oxidase B (MAO-B) catalyzes the oxidation of amines and is inhibited for the treatment of both Parkinson's disease and depression. Despite its clinical importance, its catalytic mechanism remains unclear, and routes to drugging this target would be valuable. Evidence of a radical in either the transition state or the resting state of MAO-B is present throughout the literature and is suggested to be a flavin semiquinone, a tyrosyl radical, or both. Here we see evidence of a resting-state flavin semiquinone, via absorption redox studies and electron paramagnetic resonance, suggesting that the anionic semiquinone is biologically relevant. On the basis of enzyme kinetic studies, enzyme variants, and molecular dynamics simulations, we find evidence for the importance of the membrane environment in mediating the activity of MAO-B and that this mediation is related to the protein dynamics of MAO-B. Further, our MD simulations identify a hitherto undescribed entrance for substrate binding, membrane modulated substrate access, and indications for half-site reactivity: only one active site is accessible to binding at a time. Our study combines both experimental and computational evidence to illustrate the subtle interplay between enzyme activity and protein dynamics and the immediate membrane environment. Understanding key biomedical enzymes to this level of detail will be crucial to inform strategies (and binding sites) for rational drug design for these targets.

Topics: Binding Sites; Catalytic Domain; Cell Membrane; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Humans; Kinetics; Molecular Dynamics Simulation; Monoamine Oxidase; Oxidation-Reduction; Protein Binding

Flavodoxins are small proteins with a non-covalently bound FMN that can accept two electrons and accordingly adopt three redox states: oxidized (quinone), one-electron reduced (semiquinone), and two-electron reduced (quinol). In iron-deficient cyanobacteria and algae, flavodoxin can substitute for ferredoxin as the electron carrier in the photosynthetic electron transport chain. Here, we demonstrate a similar function for flavodoxin from the green sulfur bacterium Chlorobium phaeovibrioides (cp-Fld). The expression of the cp-Fld gene, found in a close proximity with the genes for other proteins associated with iron transport and storage, increased in a low-iron medium. cp-Fld produced in Escherichia coli exhibited the optical, ERP, and electron-nuclear double resonance spectra that were similar to those of known flavodoxins. However, unlike all other flavodoxins, cp-Fld exhibited unprecedented stability of FMN semiquinone to oxidation by air and difference in midpoint redox potentials for the quinone-semiquinone and semiquinone-quinol couples (- 110 and - 530 mV, respectively). cp-Fld could be reduced by pyruvate:ferredoxin oxidoreductase found in the membrane-free extract of Chl. phaeovibrioides cells and photo-reduced by the photosynthetic reaction center found in membrane vesicles from these cells. The green sulfur bacterium Chl. phaeovibrioides appears thus to be a new type of the photosynthetic organisms that can use flavodoxin as an alternative electron carrier to cope with iron deficiency.

Topics: Air; Chlorobi; Electron Spin Resonance Spectroscopy; Electrons; Escherichia coli; Flavin-Adenine Dinucleotide; Flavodoxin; Oxidation-Reduction; Pyruvate Synthase

The redox-neutral reaction catalyzed by 2-haloacrylate hydratase (2-HAH) leads to the conversion of 2-chloroacrylate to pyruvate. Previous mechanistic studies demonstrated the formation of a flavin-iminium ion as an important intermediate in the 2-HAH catalytic cycle. Time-resolved flavin absorbance studies were performed in this study, and the data showed that the enzyme is capable of stabilizing both anionic and neutral flavin semiquinone species. The presence of a radical scavenger decreases the activity in a concentration-dependent manner. These data are consistent with the flavin iminium intermediate occurring by radical recombination.

Topics: Acrylates; Bacteria; Flavin-Adenine Dinucleotide; Flavins; Flavoproteins; NADP; Oxidation-Reduction; Pyruvic Acid

Formate oxidase (FOX) was previously shown to contain a noncovalently bound 8-formyl FAD (8-fFAD) cofactor. However, both the absorption spectra and the kinetic parameters previously reported for FOX are inconsistent with more recent reports. The ultraviolet-visible (UV-vis) absorption spectrum reported in early studies closely resembles the spectra observed for protein-bound 8-formyl flavin semiquinone species, thus suggesting FOX may be photosensitive. Therefore, the properties of dark and light-exposed FOX were investigated using steady-state kinetics and site-directed mutagenesis analysis along with inductively coupled plasma optical emission spectroscopy, UV-vis absorption spectroscopy, circular dichroism spectroscopy, liquid chromatography and mass spectrometry, and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, these experimental results demonstrate that FOX is deactivated in the presence of light through generation of an oxygen stable, anionic (red) 8-fFAD semiquinone radical capable of persisting either in an aerobic environment for multiple weeks or in the presence of a strong reducing agent like sodium dithionite. Herein, we study the photoinduced formation of the 8-fFAD semiquinone radical in FOX and report the first EPR spectrum of this radical species. The stability of the 8-fFAD semiquinone radical suggests FOX to be a model enzyme for probing the structural and mechanistic features involved in stabilizing flavin semiquinone radicals. It is likely that the photoinduced formation of a stable 8-fFAD semiquinone radical is a defining characteristic of 8-formyl flavin-dependent enzymes. Additionally, a better understanding of the radical stabilization process may yield a FOX enzyme with more robust activity and broader industrial usefulness.

Topics: Aspergillus; Benzoquinones; Flavin-Adenine Dinucleotide; Fungal Proteins; Mutagenesis, Site-Directed; Oxidoreductases; Ultraviolet Rays

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

Topics: Binding Sites; Electron Transport; Electrons; Flavin-Adenine Dinucleotide; Flavins; Models, Biological

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (

Topics: Apoenzymes; Bacterial Proteins; Benzoic Acid; Biocatalysis; Desulfovibrio vulgaris; Electron Transport; Enterobacter cloacae; Flavin-Adenine Dinucleotide; Flavodoxin; Holoenzymes; Models, Molecular; Multienzyme Complexes; NADH, NADPH Oxidoreductases; Nitroreductases; ortho-Aminobenzoates; Oxidation-Reduction; Oxidoreductases; Pyrococcus furiosus; Recombinant Fusion Proteins; Recombinant Proteins; Silent Mutation; Thermus thermophilus

Flavin has long been known to function as a single electron reductant in biological settings, but this reactivity has rarely been observed with flavoproteins used in organic synthesis. Here we describe the discovery of an enantioselective radical dehalogenation pathway for α-bromoesters using flavin-dependent 'ene'-reductases. Mechanistic experiments support the role of flavin hydroquinone as a single electron reductant, flavin semiquinone as the hydrogen atom source, and the enzyme as the source of chirality.

Topics: Electron Transport; Esters; Flavin-Adenine Dinucleotide; Flavins; Gluconobacter oxydans; Halogenation; Hydrogen; Models, Molecular; NADP; Oxidation-Reduction; Oxidoreductases; Stereoisomerism

Short-lived radicals generated in the photoexcitation of flavin adenine dinucleotide (FAD) in aqueous solution at low pH are detected with high sensitivity and spatial resolution using a newly developed transient optical absorption detection (TOAD) imaging microscope. Radicals can be studied under both flash photolysis and continuous irradiation conditions, providing a means of directly probing potential biological magnetoreception within sub-cellular structures. Direct spatial imaging of magnetic field effects (MFEs) by magnetic intensity modulation (MIM) imaging is demonstrated along with transfer and inversion of the magnetic field sensitivity of the flavin semiquinone radical concentration to that of the ground state of the flavin under strongly pumped reaction cycling conditions. A low field effect (LFE) on the flavin semiquinone-adenine radical pair is resolved for the first time, with important implications for biological magnetoreception through the radical pair mechanism.

Topics: Flavin-Adenine Dinucleotide; Free Radicals; Magnetic Fields; Microscopy; Photochemical Processes; Photolysis

Since cyanide potentiates the inhibitory activity of several monoamine oxidase (MAO) inhibitors, a series of carbonitrile-containing aminoheterocycles was examined to explore the role of nitriles in determining the inhibitory activity against MAO. Dicarbonitrile aminofurans were found to be potent, selective inhibitors against MAO A. The origin of the MAO A selectivity was identified by combining spectroscopic and computational methods. Spectroscopic changes induced in MAO A by mono- and dicarbonitrile inhibitors were different, providing experimental evidence for distinct binding modes to the enzyme. Similar differences were also found between the binding of dicarbonitrile compounds to MAO A and to MAO B. Stabilization of the flavin anionic semiquinone by monocarbonitrile compounds, but destabilization by dicarbonitriles, provided further support to the distinct binding modes of these compounds and their interaction with the flavin ring. Molecular modeling studies supported the role played by the nitrile and amino groups in anchoring the inhibitor to the binding cavity. In particular, the results highlight the role of Asn181 and Ile335 in assisting the interaction of the nitrile-containing aminofuran ring. The network of interactions afforded by the specific attachment of these functional groups provides useful guidelines for the design of selective, reversible MAO A inhibitors.

Topics: Asparagine; Binding Sites; Flavin-Adenine Dinucleotide; Furans; Humans; Isoleucine; Models, Molecular; Molecular Docking Simulation; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Protein Interaction Domains and Motifs; Spectrum Analysis; Structure-Activity Relationship

Extracellular redox-active compounds, flavins and other quinones, have been hypothesized to play a major role in the delivery of electrons from cellular metabolic systems to extracellular insoluble substrates by a diffusion-based shuttling two-electron-transfer mechanism. Here we show that flavin molecules secreted by Shewanella oneidensis MR-1 enhance the ability of its outer-membrane c-type cytochromes (OM c-Cyts) to transport electrons as redox cofactors, but not free-form flavins. Whole-cell differential pulse voltammetry revealed that the redox potential of flavin was reversibly shifted more than 100 mV in a positive direction, in good agreement with increasing microbial current generation. Importantly, this flavin/OM c-Cyts interaction was found to facilitate a one-electron redox reaction via a semiquinone, resulting in a 10(3)- to 10(5)-fold faster reaction rate than that of free flavin. These results are not consistent with previously proposed redox-shuttling mechanisms but suggest that the flavin/OM c-Cyts interaction regulates the extent of extracellular electron transport coupled with intracellular metabolic activity.

Topics: Biofilms; Cytochrome c Group; Cytochromes c; Electrochemistry; Electrodes; Electron Spin Resonance Spectroscopy; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Heme; Microscopy, Confocal; Nucleotides; Oxidation-Reduction; Shewanella

Topics: Cytochrome c Group; Electron Transport; Flavin-Adenine Dinucleotide; Shewanella

One crucial component in light signaling is the quantity of photoreceptor present in the active signaling state. The lifetime of the signaling state of a photoreceptor is limited because of thermal or otherwise back reversion of the chromophore to the ground state, and/or degradation of the photoreceptor in the light-activated state. It was previously shown that the lit state of plant cryptochromes contains flavin-neutral semiquinone, and that the half-lives of the lit state were in the range of 3-4 min in vitro. However, it was unknown how long-lived the signaling states of plant cryptochromes are in situ. Based on the loss of degradation of cry2 after prolonged dark incubation and loss of reversibility of photoactivated cry1 by a pulse of green light, we estimate the in vivo half-lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively. Based on electron paramagnetic resonance measurements, the lifetime of the Arabidopsis cry1 lit state in insect cells was found to be ~6 min, and thus very similar to the lifetime of the signaling state in planta. Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Cell Line; Cryptochromes; Flavin-Adenine Dinucleotide; Gene Expression Regulation, Plant; Genes, Reporter; Insecta; Light; Light Signal Transduction; Mutation; Photoreceptors, Plant; Plants, Genetically Modified; Seedlings; Time Factors

The reactions of the flavin semiquinone generated by laser-induced stepwise two-photon excitation of reduced flavin have been studied previously (El Hanine-Lmoumene C & Lindqvist L. (1997) Photochem Photobiol 66, 591-595) using time-resolved spectroscopy. In the present work, we have used the same experimental procedure to study the flavin semiquinone in rat kidney long-chain hydroxy acid oxidase and in the flavodehydrogenase domain of flavocytochrome b(2) FDH, two homologous flavoproteins belonging to the family of FMN-dependent L-2-hydroxy acid-oxidizing enzymes. For both proteins, pulsed laser irradiation at 355 nm of the reduced enzyme generated initially the neutral semiquinone, which has rarely been observed previously for these enzymes, and hydrated electron. The radical evolved with time to the anionic semiquinone that is known to be stabilized by these enzymes at physiological pH. The deprotonation kinetics were biphasic, with durations of 1-5 micros and tens of microseconds, respectively. The fast phase rate increased with pH and Tris buffer concentration. However, this increase was about 10-fold less pronounced than that reported for the neutral semiquinone free in aqueous solution. pK(a) values close to that of the free flavin semiquinone were obtained from the transient protolytic equilibrium at the end of the fast phase. The second slow deprotonation phase may reflect a conformational relaxation in the flavoprotein, from the fully reduced to the semiquinone state. The anionic semiquinone is known to be an intermediate in the flavocytochrome b(2) catalytic cycle. In light of published kinetic studies, our results indicate that deprotonation of the flavin radical is not rate-limiting for the intramolecular electron transfer processes in this protein.

Topics: Alcohol Oxidoreductases; Animals; Flavin-Adenine Dinucleotide; Hydroxy Acids; Kidney; Kinetics; L-Lactate Dehydrogenase (Cytochrome); Lasers; Photolysis; Rats; Spectrum Analysis

The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O(2) absent) or > 2 (O(2) present), the apparent K (M) value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.

Topics: Bacterial Proteins; Chromates; Flavin-Adenine Dinucleotide; Flavoproteins; FMN Reductase; Hydrogen-Ion Concentration; NAD; Oxidation-Reduction; Oxidative Stress; Oxidoreductases; Oxygen Consumption; Paracoccus denitrificans

Photolyases and cryptochromes (CRY) are structurally homologous flavoproteins with divergent functions. While photolyases repair UV-damaged DNA by photoinduced electron transfer from their FAD cofactor, CRY are involved in varied cellular processes, including light-dependent plant growth, regulation of mammalian circadian rhythm, and possibly magnetoreception. Despite their importance in Nature and human health, little is known about how they tune their FAD redox properties to achieve remarkable functional diversity. In this study, we reveal a kinetic mechanism, exploited by cyclobutane pyrimidine dimer photolyase (PL), for regulating the stability of its FAD semiquinone (sq). We find that the sq in CRY-DASH (Synechocystis) is substantially more reactive toward oxidation than in PL (Anacystis nidulans) and, using deuterium isotope and pH effects, show that rate-limiting proton transfer contributes to the exceptional kinetic stability of the PL sq. Through mutagenesis, we identify two PL-specific residues in the flavin binding pocket, Trp392 and Gly389 (Try398 and Asn395 in CRY-DASH, respectively), that ensure this kinetic stability, possibly through interactions with the adenine moiety of FAD and/or adjusting the polarity of the binding site. Significantly, these relatively distal residues have a much more profound impact than two amino acids closer to the FAD. By quantifying sq stability in a series of PL-CRY exchange mutants, our findings pave the way for investigations aimed at correlating sq stability with function in these proteins. As is being recognized with other flavoproteins, we expect that kinetic tuning of the rates of electron transfer will play a function-defining role in photolyases and cryptochromes.

Topics: Aspartic Acid; Binding Sites; Cryptochromes; Crystallography, X-Ray; Deoxyribodipyrimidine Photo-Lyase; Dimerization; Electron Transport; Flavin-Adenine Dinucleotide; Glycine; Hydrogen-Ion Concentration; Kinetics; Osmolar Concentration; Oxidation-Reduction; Tryptophan

Histamine dehydrogenase from Nocardioides simplex is a homodimer and belongs to the family of iron-sulfur flavoproteins having one [4Fe-4S] cluster and one 6-S-cysteinyl FMN per monomer. In the reductive titration with histamine, two-electron reduction occurred per monomer at pH<9, while single-electron reduction proceeded at pH>9. The substrate-reduced histamine dehydrogenase yielded an electron paramagnetic resonance spectral signal assigned to the flavin semiquinone. The signal intensity increased with pH up to pH 9 and reached a maximum at pH>9. These unique features are explained in terms of the redox potential of the cofactors, where the redox potential was evaluated over a pH range from 7 to 10 by using a spectroelectrochemical titration method for the flavin and cyclic voltammetry for the [4Fe-4S] cluster. The bell-type pH dependence of the enzymatic activity is also discussed in terms of the pH dependence of the centers' redox potential.

Topics: Electron Spin Resonance Spectroscopy; Electron Transport; Flavin-Adenine Dinucleotide; Histamine; Hydrogen-Ion Concentration; Iron-Sulfur Proteins; Nocardiaceae; Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Thermodynamics

Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH(.)) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH(.) (whereas catalytically active photolyase requires fully reduced flavin (FADH(-))) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.

Topics: Arabidopsis; Arabidopsis Proteins; Coenzymes; Cryptochromes; DNA Repair; DNA, Single-Stranded; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Flavoproteins; Light; Oxidation-Reduction; Pyrimidine Dimers; Signal Transduction; Spectrophotometry, Ultraviolet

Saccharomyces cerevisiae flavocytochrome b2 (L-lactate:cytochrome c oxido reductase, EC 1.1.2.3) is a homotetramer, with FMN and protoheme IX binding on separate domains. The flavin-binding domains form the enzyme tetrameric core, while the cytochrome b2 domains appear to be mobile around a hinge region (Xia, Z. X., and Mathews, F. S. (1990) J. Mol. Biol. 212, 867-863). The enzyme catalyzes electron transfer from L-lactate to cytochrome c, or to nonphysiological acceptors such as ferricyanide, via FMN and heme b2. The kinetics of this multistep reaction are complex. In order to clarify some of its aspects, the tetrameric FMN-binding domain (FDH domain) has been independently expressed in Escherichia coli (Balme, A., Brunt, C. E., Pallister, R., Chapman, S. K., and Reid, G. A. (1995) Biochem. J. 309, 601-605). We present here an additional characterization of this domain. In our hands, it has essentially the same ferricyanide reductase activity as the holo-enzyme. The comparison of the steady-state kinetics with ferricyanide as acceptor and of the pre-steady-state kinetics of flavin reduction, as well as the kinetic isotope effects of the reactions using L-2-[2H]lactate, indicates that flavin reduction is the limiting step in lactate oxidation. During the oxidation of the reduced FDH domain by ferricyanide, the oxidation of the semiquinone is much faster than the oxidation of two-electron-reduced flavin. This order of reactivity is reversed during flavin to heme b2 transfer in the holo-enzyme. Potentiometric studies of the protein yielded a standard redox potential for FMN at pH 7.0, E(o)7, of -81 mV, a value practically identical to the published value of -90 mV for FMN in holo-flavocytochrome b2. However, as expected from the kinetics of the oxidative half-reaction, the FDH domain was characterized by a significantly destabilized flavin semiquinone state compared with holo-enzyme, with a semiquinone formation constant K of 1.25-0.64 vs 33.5, respectively (Tegoni, M., Silvestrini, M. C., Guigliarelli, B., Asso, M., and Bertrand, P. (1998) Biochemistry, 37, 12761-12771). As in the holo-enzyme, the semiquinone state in the FDH domain is significantly stabilized by the reaction product, pyruvate. We also studied the inhibition exerted in the steady and pre steady states by the reaction product pyruvate and by anions (bromide, chloride, phosphate, acetate), with respect to both flavin reduction and reoxidation. The results indicate that these compounds bind t

Topics: Algorithms; Anions; Binding Sites; Ferricyanides; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavins; Kinetics; L-Lactate Dehydrogenase (Cytochrome); Models, Chemical; Models, Molecular; NADH, NADPH Oxidoreductases; Oxidation-Reduction; Protein Binding; Protein Structure, Tertiary; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

4-Hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum catalyses the reversible dehydration of its substrate 4-hydroxybutyryl-CoA (4-HB-CoA) to crotonyl CoA. The enzyme contains one [4Fe-4S](2+) cluster and one flavin adenine dinucleotide (FAD) molecule per homotetramer. Incubation of the enzyme with its substrate under equilibrium conditions followed by freezing at 77K induced the EPR-spectrum of a neutral flavin semiquinone (g=2.005, linewidth 2.1 mT), while at 10K additional signals were detected. In an attempt to characterize these signals, 4-HB-CoA molecules specifically labeled with (13)C have been synthesized. This was achieved via (13)C-labeled gamma-butyrolactones, which were obtained from (13)C-labeled bromoacetic acids by efficient synthetic routes. Incubation of the (13)C-labeled 4-hydroxybutyrate-CoA molecules with 4-hydroxybutyryl-CoA dehydratase did not lead to marked broadening of the signals.

Topics: Acyl Coenzyme A; Carbon Isotopes; Catalysis; Clostridium; Dehydration; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Hydro-Lyases; Models, Chemical; Sodium Oxybate

Cyclobutane-type pyrimidine dimers generated by ultraviolet irradiation of DNA can be cleaved by DNA photolyase. The enzyme-catalysed reaction is believed to be initiated by the light-induced transfer of an electron from the anionic FADH- chromophore of the enzyme to the pyrimidine dimer. In this contribution, first infrared experiments using a novel E109A mutant of Escherichia coli DNA photolyase, which is catalytically active but unable to bind the second cofactor methenyltetrahydrofolate, are described. A stable blue-coloured form of the enzyme carrying a neutral FADH radical cofactor can be interpreted as an intermediate analogue of the light-driven DNA repair reaction and can be reduced to the enzymatically active FADH- form by red-light irradiation. Difference Fourier transform infrared (FT-IR) spectroscopy was used to monitor vibronic bands of the blue radical form and of the fully reduced FADH- form of the enzyme. Preliminary band assignments are based on experiments with 15N-labelled enzyme and on experiments with D2O as solvent. Difference FT-IR measurements were also used to observe the formation of thymidine dimers by ultraviolet irradiation and their repair by light-driven photolyase catalysis. This study provides the basis for future time-resolved FT-IR studies which are aimed at an elucidation of a detailed molecular picture of the light-driven DNA repair process.

Topics: Bacillus subtilis; Catalysis; Deoxyribodipyrimidine Photo-Lyase; DNA Damage; DNA Repair; Enzyme Activation; Escherichia coli; Flavin-Adenine Dinucleotide; Folic Acid; Light; Mutation; Photochemistry; Spectroscopy, Fourier Transform Infrared; Thymine; Uridine

Flavoenzymes may reduce quinones in a single-electron, mixed single- and two-electron, and two-electron way. The mechanisms of two-electron reduction of quinones are insufficiently understood. To get an insight into the role of flavin semiquinone stability in the regulation of single- vs. two-electron reduction of quinones, we studied the reactions of wild type Anabaena ferredoxin:NADP(+)reductase (FNR) with 48% FAD semiquinone (FADH*) stabilized at the equilibrium (pH 7.0), and its Glu301Ala mutant (8% FADH* at the equilibrium). We found that Glu301Ala substitution does not change the quinone substrate specificity of FNR. However, it confers the mixed single- and two-electron mechanism of quinone reduction (50% single-electron flux), whereas the wild type FNR reduces quinones in a single-electron way. During the oxidation of fully reduced wild type FNR by tetramethyl-1,4-benzoquinone, the first electron transfer (formation of FADH*) is about 40 times faster than the second one (oxidation of FADH*). In contrast, the first and second electron transfer proceeded at similar rates in Glu301Ala FNR. Thus, the change in the quinone reduction mechanism may be explained by the relative increase in the rate of second electron transfer. This enabled us to propose the unified scheme of single-, two- and mixed single- and two-electron reduction of quinones by flavoenzymes with the central role of the stability of flavin/quinone ion-radical pair.

Topics: Alanine; Amino Acid Substitution; Anabaena; Electrons; Ferredoxin-NADP Reductase; Flavin-Adenine Dinucleotide; Glutamic Acid; Hydrogen-Ion Concentration; Kinetics; Oxidation-Reduction; Quinones

The reduction potential of the (FADH-/FADH*) couple in DNA photolyase was measured, and the value was found to be significantly higher than the values estimated in the literature. In the absence of substrate, the enzyme has a reduction potential of 16 +/- 6 mV vs NHE. In the presence of excess substrate the reduction potential increases to 81 +/- 8 mV vs NHE. The increase in reduction potential has physiological relevance since it gives the catalytic state greater resistance to oxidation. This is the first measurement of a reduction potential for this class of DNA-repair enzymes and the larger family of blue-light photoreceptors.

Topics: Catalysis; Deoxyribodipyrimidine Photo-Lyase; DNA Repair; Electrochemistry; Enzyme Stability; Flavin-Adenine Dinucleotide; Kinetics; Oxidation-Reduction; Pyrimidine Dimers

2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase (MHPCO) is a flavoprotein that catalyzes the oxygenation of MHPC to form alpha-(N-acetylaminomethylene)-succinic acid. Although formally similar to the oxygenation reactions catalyzed by phenol hydroxylases, MHPCO catalyzes the oxygenation of a pyridyl derivative rather than a simple phenol. Therefore, in this study, the mechanism of the reaction was investigated by replacing the natural cofactor FAD with FAD analogues having various substituents (-Cl, -CN, -NH(2), -OCH(3)) at the C8-position of the isoalloxazine. Thermodynamic and catalytic properties of the reconstituted enzyme were investigated and found to be similar to those of the native enzyme, validating that these FAD analogues are reasonable to be used as mechanistic probes. Dissociation constants for the binding of MHPC or the substrate analogue 5-hydroxynicotinate (5HN) to the reconstituted enzymes indicate that the reconstituted enzymes bind well with ligands. Redox potential values of the reconstituted enzymes were measured and found to be more positive than the values of free FAD analogues, which correlated well with the electronic effects of the 8-substituents. Studies of the reductive half-reaction of MHPCO have shown that the rates of flavin reduction by NADH could be described as a parabolic relationship with the redox potential values of the reconstituted enzymes, which is consistent with the Marcus electron transfer theory. Studies of the oxidative half-reaction of MHPCO revealed that the rate of hydroxylation depended upon the different analogues employed. The rate constants for the hydroxylation step correlated with the calculated pK(a) values of the 8-substituted C(4a)-hydroxyflavin intermediates, which are the leaving groups in the oxygen transfer step. It was observed that the rates of hydroxylation were greater when the pK(a) values of C(4a)-hydroxyflavins were lower. Although these results are not as dramatic as those from analogous studies with parahydroxybenzoate hydroxylase (Ortiz-Maldonado et al., (1999) Biochemistry 38, 8124-8137), they are consistent with the model that the oxygenation reaction of MHPCO occurs via an electrophilic aromatic substitution mechanism analogous to the mechanisms for parahydroxybenzoate and phenol hydroxylases.

Topics: Apoenzymes; Binding Sites; Catalysis; Flavin-Adenine Dinucleotide; Flavoproteins; Hydroxylation; Kinetics; Mixed Function Oxygenases; Nicotinic Acids; Oxidation-Reduction; Oxygen; Spectrophotometry, Ultraviolet; Substrate Specificity; Thermodynamics

In this paper, we report a spectroelectrochemical investigation of proton-coupled electron transfer in flavodoxin D. vulgaris Hildenborough (Fld). Poly-L-lysine is used to promote the binding of Fld to the nanocrystalline, mesoporous SnO(2) electrodes. Two reversible redox couples of the immobilized Fld are observed electrochemically and are assigned by spectroelectrochemistry to the quinone/semiquinone and semiquinone/hydroquinone couples of the protein's flavin mononucleotide (FMN) redox cofactor. Comparison with control data for free FMN indicates no contamination of the Fld data by dissociated FMN. The quinone/semiquinone and semiquinone/hydroquinone midpoint potentials (E(q/sq) and E(sq/hq)) at pH 7 were determined to be -340 and -585 mV vs Ag/AgCl, in good agreement with the literature. E(q/sq) exhibited a pH dependence of 51 mV/pH. The kinetics of these redox couples were studied using cyclic voltammetry, cyclic voltabsorptometry, and chronoabsorptometry. The semiquinone/quinone reoxidation is found to exhibit slow, potential-independent but pH-sensitive kinetics with a reoxidation rate constant varying from 1.56 s(-)(1) at pH 10 to 0.0074 s(-)(1) at pH 5. The slow kinetics are discussed in terms of a simple kinetics model and are assigned to the reoxidation process being rate limited by semiquinone deprotonation. It is proposed that this slow deprotonation step has the physiological benefit of preventing the undesirable loss of reducing equivalents which results from semiquinone oxidation to quinone.

Topics: Crystallization; Electrodes; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavodoxin; Hydrogen-Ion Concentration; Hydroquinones; Kinetics; Microchemistry; Oxidation-Reduction; Polylysine; Protons; Quinones; Thermodynamics; Tin Compounds

Neuronal nitric-oxide synthase (nNOS) differs from inducible NOS (iNOS) in both its dependence on the intracellular Ca2+ concentration and the production rate of NO. To investigate what difference(s) exist between the two NOS flavin domains at the electron transfer level, we isolated the recombinant human NOS flavin domains, which were co-expressed with human calmodulin (CaM). The flavin semiquinones, FADH* and FMNH*, in both NOSs participate in the regulation of one-electron transfer within the flavin domain. Each semiquinone can be identified by a characteristic absorption peak at 520 nm (Guan, Z.-W., and Iyanagi, T. (2003) Arch. Biochem. Biophys. 412, 65-76). NADPH reduction of the FAD and FMN redox centers by the CaM-bound flavin domains was studied by stopped-flow and rapid scan spectrometry. Reduction of the air-stable semiquinone (FAD-FMNH*) of both domains with NADPH showed that the extent of conversion of FADH2/FMNH* to FADH*/FMNH2 in the iNOS flavin domain was greater than that of the nNOS flavin domain. The reduction of both oxidized domains (FAD-FMN) with NADPH resulted in the initial formation of a small amount of disemiquinone, which then decayed. The rate of intramolecular electron transfer between the two flavins in the iNOS flavin domain was faster than that of the nNOS flavin domain. In addition, the formation of a mixture of the two- and four-electron-reduced states in the presence of excess NADPH was different for the two NOS flavin domains. The data indicate a more favorable formation of the active intermediate FMNH2 in the iNOS flavin domain.

Topics: Calmodulin; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Humans; Kinetics; NADP; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Oxidation-Reduction; Protein Structure, Tertiary

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.

Topics: Alcohol Oxidoreductases; Arthrobacter; Betaine; Catalysis; Choline; Electron Spin Resonance Spectroscopy; Enzyme Inhibitors; Escherichia coli; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Kinetics; Recombinant Proteins; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

The extracellular enzymatic reduction of iron by microorganisms has not been appropriately considered. In this study the reduction and release of iron from ferrioxamine were examined using extracellular microbial iron reductases and compared to iron mobilization by chemical reductants, and to chelation by EDTA and desferrioxamine. A flavin semiquinone was formed during the enzymatic reduction of ferrioxamine, which was consistent with the 1 e(-) reduction of iron by an enzyme. The rates for the enzymatic reactions were substantially faster than both the 2 e(-) chemical reductions and the chelation reactions. The rapid rates of the enzymatic reduction reactions demonstrated that these enzymes are capable of accomplishing the extracellular mobilization of iron required by microorganisms. The data suggest that mechanistically there are two phases for the mobilization and transport of iron by those microorganisms that produce both extracellular iron reductases and siderophores, with reduction being the principle pathway.

Topics: Bacteria; Bacterial Proteins; Biological Transport; Deferoxamine; Escherichia coli; Extracellular Space; Ferric Compounds; Flavin-Adenine Dinucleotide; Iron; Iron Chelating Agents; Kinetics; Listeria monocytogenes; Oxidation-Reduction; Oxidoreductases; Pseudomonas aeruginosa; Spectrophotometry; Transferrin; Yersinia enterocolitica

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mössbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Topics: Catalysis; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Iron-Sulfur Proteins; Molybdenum; Oxidation-Reduction; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Spectroscopy, Mossbauer; Xanthine Oxidase

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.

Topics: 2,6-Dichloroindophenol; Buffers; Catalysis; Decarboxylation; Deuterium Oxide; Flavin-Adenine Dinucleotide; Kinetics; Lactobacillus; Oxidation-Reduction; Phosphates; Potassium Compounds; Pyruvate Oxidase; Pyruvic Acid; Solvents; Spectrophotometry; Substrate Specificity

Bromonitromethane is an inefficient suicide substrate for glucose oxidase (in contrast to the case of CH(3)CCl=NO(2)(-) and D-amino acid oxidase) because, in the enzyme-substrate encounter step, the required ionization states of enzyme (EH(0)(+), pK(a) approximately 3.5) and substrate (CHBr=NO(2)(-), pK(a) approximately 8.3) cannot be highly populated simultaneously. Because reprotonation of CHBr=NO(2)(-) is rapid at the pH value used for the assay of glucose oxidase, presentation of the enzyme with the preformed anion could not be exploited in this case. We circumvent this difficulty by allowing the enzyme to reductively dehalogenate CHBr(2)NO(2), thereby generating the desired protonically unstable suicide substrate in situ (E(r) + CHBr(2)NO(2) --> E(o) + CHBr=NO(2)(-) + HBr + H(+)). Irreversible inactivation of the enzyme, because of the formation of a dead-end N-5 formylflavin adduct, is more than 100-fold faster when CHBr=NO(2)(-) is generated in situ than when it is externally applied. The remaining competitive fates of CHBr=NO(2)(-) at the active site are protonation and release or oxidation to HCOBr (or HCONO(2)). Strong support for these conclusions comes from (1) the brisk evolution of CH(3)CBr=NO(2)(-) (which is too bulky to act further as an efficient suicide substrate) from the enzyme-catalyzed reductive debromination of CH(3)CBr(2)NO(2), (2) the 1:1 stoichiometry of enzyme inactivation, and (3) the identification of the modified flavin as 5-formyl-1, 5-dihydro-FAD.

Topics: Anions; Aspergillus niger; Binding Sites; Bromine; Dioxygenases; Enzyme Inhibitors; Enzyme Precursors; Flavin-Adenine Dinucleotide; Glucose; Glucose Oxidase; Kinetics; Methane; Nitroparaffins; Oxygenases; Protons; Structure-Activity Relationship; Substrate Specificity

DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.

Topics: Deoxyribodipyrimidine Photo-Lyase; Electron Spin Resonance Spectroscopy; Escherichia coli; Flavin-Adenine Dinucleotide; Hot Temperature; Oxidation-Reduction; Spin Labels; Tryptophan

The influence of the amino acid residues surrounding the flavin ring in the flavodoxin of the cyanobacterium Anabaena PCC 7119 on the electron spin density distribution of the flavin semiquinone was examined in mutants of the key residues Trp(57) and Tyr(94) at the FMN binding site. Neutral semiquinone radicals of the proteins were obtained by photoreduction and examined by electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies. Significant differences in electron density distribution were observed in the flavodoxin mutants Trp(57) --> Ala and Tyr(94) --> Ala. The results indicate that the presence of a bulky residue (either aromatic or aliphatic) at position 57, as compared with an alanine, decreases the electron spin density in the nuclei of the benzene flavin ring, whereas an aromatic residue at position 94 increases the electron spin density at positions N(5) and C(6) of the flavin ring. The influence of the FMN ribityl and phosphate on the flavin semiquinone was determined by reconstituting apoflavodoxin samples with riboflavin and with lumiflavin. The coupling parameters of the different nuclei of the isoalloxazine group, as detected by ENDOR and HYSCORE, were very similar to those of the native flavodoxin. This indicates that the protein conformation around the flavin ring and the electron density distribution in the semiquinone form are not influenced by the phosphate and the ribityl of FMN.

Topics: Amino Acid Substitution; Anabaena; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Flavins; Flavodoxin; Hydrogen; Point Mutation; Riboflavin

The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent. While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic. The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable. As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins. This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine. This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered. Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed. The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7). The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type. Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone. On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant. The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple. On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.

Topics: Circular Dichroism; Clostridium; Coenzymes; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavodoxin; Glutamic Acid; Glutamine; Hydrogen Bonding; Hydrogen-Ion Concentration; Hydroquinones; Mutagenesis, Site-Directed; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Protein Binding; Protons; Spectrophotometry, Ultraviolet; Temperature; Thermodynamics

Topics: Catalysis; Electron Spin Resonance Spectroscopy; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Flavoproteins; Glucose Oxidase; Hydroquinones; Oxidation-Reduction; Quinones

Nitric oxide synthase (NOS) is a heme protein that catalyzes the oxygenation of L-arginine in the presence of NADPH to form nitric oxide, L-citrulline and NADP+, and proceeds via two partial reactions: 1) L-Arginine --> NG-hydroxy-L-arginine 2) NG-Hydroxy-L-arginine --> L-citrulline + nitric oxide Calmodulin, FAD, FMN and tetrahydrobiopterin are required for both reactions. Reactions 1 and 2 require the input of 2 and 1 electron equivalents, respectively. Under normal multiple turnover conditions, these electrons are ultimately derived from NADPH. We previously reported that NOS contains an endogenous reductant that, in the absence of NADPH, can support the single-turnover oxygenation of L-arginine to NG-hydroxy-L-arginine and a relatively small amount of L-citrulline [Campos, K. L., Giovanelli, J., and Kaufman, S. (1995) J. Biol. Chem. 270, 1721-1728]. This reductant has now been identified as the stable flavin semiquinone free radical (FSQ). Its oxidation appears to be coupled to the formation of NG-hydroxy-L-arginine and L-citrulline. The rate of FSQ oxidation is two orders of magnitude slower than the flux of electrons from NADPH through NOS during normal turnover of the enzyme, indicating that FSQ is not the proximal electron donor for heme under these conditions.

Topics: Arginine; Citrulline; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Kinetics; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Oxygen

The electron spin echo envelope modulation (ESEEM) technique of pulsed EPR spectroscopy was applied to the anionic semiquinone of the cholesterol oxidase flavin cofactor, formed when the enzyme was photoreduced in the presence of 5-deazariboflavin and EDTA. Fourier transforms of the three-pulse ESEEM spectra showed the presence of 14N nuclei magnetically coupled to the paramagnet. In 2H2O buffer the surroundings of the flavin ring were shown to be accessible to solvent exchange, with a deuterium population in close proximity to the paramagnetic centre. Upon binding of the pseudosubstrate, dehydroisoandrosterone, subtle changes were observed in the coupling to nitrogen nuclei, which are interpreted as changes in the electron density distribution of the flavin ring system. The results are discussed in terms of the three-dimensional structure reported for the protein and the flavin ring architecture.

Topics: Bacterial Proteins; Benzoquinones; Brevibacterium; Cholesterol Oxidase; Dehydroepiandrosterone; Deuterium Oxide; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Molecular Structure

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds heme, tetrahydrobiopterin, and the substrate L-arginine. One flavin in resting nNOS exits as an air-stable semiquinone radical. During NO synthesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 HZ at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin coupling. The flavin radical, probably the semiquinone FMNH., was shown by progressive microwave power saturation and EPR saturation recovery under anaerobic conditions to be spin-spin coupled with the heme iron located in the nNOS oxygenase domain. Analysis of an nNOS preparation that was devoid of heme but contained the flavin radical revealed that spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substate analogue Nomega-nitro-L-arginine methyl ester (NAME) had no effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, and 1.81 and were unchanged by occupation of the substrate binding site by L-arginine or NAME. The substrate also had no effect on the heme zero-field splitting parameter, D=5.2cm-1. Together, the data indicate that the flavin and heme redox centers are positioned near each other in nNOS, consistent with their participating in an interdomain electron transfer. The flavin radical is affected by dissolved oxygen, suggesting that its binding site within the reductase domain partially exposed to solvent, but is unaffected when substrate binds to the oxygenase domain. Substrate binding also appears to take place outside the first coordination shell of the nNOS heme iron.

Topics: Animals; Arginine; Binding Sites; Electron Spin Resonance Spectroscopy; Electron Transport; Flavin-Adenine Dinucleotide; Free Radicals; Heme; In Vitro Techniques; Iron; Microwaves; Molecular Structure; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase

Monoamine oxidase (MAO) plays an essential role in the regulation of various neurotransmitter and xenobiotic amines. Inhibitors of MAO have been employed in the treatment of depression and as adjuncts in Parkinson's disease therapy. X-Band and Q-band electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic techniques are employed to characterize a signal assigned as a stable red anionic semiquinone radical in the resting state of MAO B. It is shown that the radical signal is not affected during substrate (either benzylamine or phenylethylamine) turnover, by anaerobic incubation with substrate, or by covalent modification of the active site flavin cofactor in the catalytically active dimer. Upon denaturation, however, the semiquinone absorbances and EPR signals are lost. Photoreduction of the native enzyme in the presence of ethylenediaminetetraacetate generates an EPR signal that is not the same as that obtained in the resting state and shows different proton ENDOR signals. These results suggest that the two flavin prosthetic groups that exist in catalytically active monoamine oxidase B are physically distinct.

Topics: Animals; Benzoquinones; Cattle; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Flavins; Liver; Models, Chemical; Molecular Structure; Monoamine Oxidase; Photochemistry; Spin Labels

4-Hydroxybutyryl-CoA dehydratase catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA, which involves cleavage of an unactivated beta-C-H bond. The enzyme also catalyzes the apparently irreversible isomerization of vinylacetyl-CoA to crotonyl-CoA. Addition of crotonyl-CoA to the dehydratase, which contains FAD as well as non-heme iron and acid labile sulfur, led to a decrease of the flavin absorbance at 438 nm and an increase in the region from 500 to 800 nm. The protein-bound FAD was easily reduced to the semiquinone (redox equilibration within seconds) and only slowly to the hydroquinone (redox equilibration minutes to hours): the redox potentials were not unusual for flavoproteins (Eox/sq = -140 +/- 15 mV and Esq/red = -240 +/- 15 mV; pH 7.0, 25 degrees C). There was no equilibration of electrons between the flavin and the Fe-S cluster, which was difficult to reduce. After extensive photoreduction, an EPR signal indicative of a [4Fe-4S]+ cluster was detected (g-values: 2.037, 1.895, 1.844). Upon exposure to air at 0 degrees C, the enzyme lost dehydration activity completely within 40 min, but isomerase activity dropped to about 40% of the initial value and persisted for more than a day. The properties of the protein-bound FAD are consistent with a mechanism involving transient one-electron oxidation of the substrate to activate the the beta-C-H bond. The putative [4Fe-4S]2+ cluster could serve a structural role and/or as Lewis acid facilitating the leaving of the hydroxyl group.

Topics: Acetyl Coenzyme A; Acyl Coenzyme A; Clostridium; Dithionite; Electron Spin Resonance Spectroscopy; Electron Transport; Flavin-Adenine Dinucleotide; Hydro-Lyases; Iron-Sulfur Proteins; Isomerism; Molecular Structure; Oxidation-Reduction; Oxygen; Photochemistry; Shikimic Acid; Spectrophotometry

Catalytically active monoamine oxidase (MAO) is believed to exist as a dimer with each subunit containing a covalently attached flavin cofactor. Fluorescence spectroscopy performed on the resting state enzyme resulted in two separate fluorescence emissions at 480 nm and 530 nm with excitation maxima at lambda ex = 412 nm and lambda ex = 450 nm, respectively. Inactivation of MAO with pargyline resulted in concomitant loss of the absorbance at 450 nm without change in the 412 nm absorption; there also was a decrease in the emission intensity at 530 nm, while the emission at 480 nm remained unchanged. The 480 nm emission decreased and the 530 nm emission intensity increased, when the enzyme was heat denatured in the presence of NaDodSO4. From these results, it is clear that there are two different chromophores present in the resting state enzyme; the 530 nm chromophore is consistent with an oxidized flavin, while the 480 nm chromophore could be a flavin semiquinone.

Topics: Animals; Cattle; Flavin-Adenine Dinucleotide; Flavins; Liver; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Oxidation-Reduction; Spectrometry, Fluorescence

The pH dependence of the behavior of chicken liver xanthine dehydrogenase in the course of reductive titrations with sodium dithionite has been examined. Below pH 8.5, the behavior of xanthine dehydrogenase is similar to that of the much better understood milk xanthine oxidase, with the amount of neutral semiquinone accumulating transiently in the course of the titration increasing somewhat as the pH decreases. At pH 10, however, an anomalously large accumulation of the neutral semiquinone is observed by both UV/visible and EPR spectroscopy. Treatment of xanthine dehydrogenase with the thiol reagent iodoacetamide significantly diminishes the ability of the enzyme to stabilize the neutral flavin semiquinone at high pH. These data are consistent with the presence of a protein thiol in the immediate vicinity of the flavin, whose ionization above pH 8.5 results in thermodynamic stabilization of the neutral flavin semiquinone over the anionic form.

Topics: Animals; Chickens; Dithionite; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Liver; Oxidation-Reduction; Spectrophotometry; Titrimetry; Xanthine Dehydrogenase

The flavoprotein monoamine oxidase B (MAO B) from bovine liver, as isolated, has an unusual additional absorption band at 412 nm, which is similar to the absorption of its anionic flavin semiquinone form, (Fl.-), and other typical (Fl.-) flavoproteins. Denaturation of the enzyme results in the elimination of this anomalous absorption. The resonance Raman (RR) spectrum of MAO B as isolated is virtually identical to that of its dithionite-reduced (Fl.-) form. Both spectra show features similar to those of the RR spectrum of the (Fl.-) form of Aspergillus niger glucose oxidase (GO) in the region between 300 and 1700 cm-1 with 406.7 nm excitation. These features are readily distinguishable from those of oxidized flavin, neutral flavin semiquinone, and hemoprotein, strongly suggesting the presence of an (Fl.-) form in MAO B as isolated, even with preparations isolated in the absence of light. There are significant differences between the RR spectra of the (Fl.-) form of MAO B and those of GO or the published RR spectra of the (Fl.-) form of D-amino acid oxidase with excess substrate analog. At least some of these differences can be attributed to the different binding of flavin in the three enzymes. No EPR signals due to (Fl.-) are observed in MAO B as isolated. The dithionite-reduced (Fl.-) form exhibits approximately 50% less EPR signal than that expected from the absorption spectrum, which suggests a possible coupling of the (Fl.-) flavin with a paramagnetic center of unknown identity in the protein. The implications of these observations on MAO B with the current view of its catalytic mechanism are discussed.

Topics: Animals; Cattle; Dithionite; Flavin-Adenine Dinucleotide; Flavoproteins; Guanidine; Guanidines; Isoenzymes; Mitochondria, Liver; Monoamine Oxidase; Protein Denaturation; Spectrophotometry; Spectrum Analysis, Raman

DNA photolyase from Escherichia coli (M(r) 54,000) consists of a polypeptide chain of 471 amino acids and the non-covalently bound cofactors methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FADH2). Two crystal forms of the enzyme were obtained; both have symmetry of space group P1. Form I has the unit cell dimensions a = 89.4 A, b = 97.3 A, c = 62.1 A, alpha = 108.3 degrees, beta = 97.4 degrees and gamma = 90.0 degrees. Diffraction from this form extends beyond 3 A resolution, but the crystals are radiation-sensitive and difficult to reproduce. Form II has the unit cell dimensions a = 62.6 A, b = 72.2 A, c = 58.5 A, alpha = 99.1 degrees, beta = 101.5 degrees and gamma = 72.0 degrees; most likely, the unit cell contains two molecules. High diffraction quality and reproducibility make form II suitable for structure analysis.

Topics: Crystallization; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Tetrahydrofolates; X-Ray Diffraction

Nitric oxide (NO) is synthesized in mammals where it acts as a signal molecule for neurotransmission, vasorelaxation, and cytotoxicity. The NO synthases isolated from brain and cytokine-activated macrophages are FAD- and FMN-containing flavoproteins that display considerable sequence homology to NADPH-cytochrome P-450 reductase. However, the nature of their catalytic centers is unknown. We have found that both isoenzymes contain 2 mol of iron-protoporphyrin IX/mol of enzyme homodimer. The optical and EPR spectroscopic properties of the heme groups were found to be remarkably similar to those of high-spin cytochrome P-450. The heme iron in the resting NO synthase is ferric and five-coordinate with a cysteine thiolate as the proximal axial ligand. In addition, the EPR spectra of the resting NO synthases contained a free radical signal attributable to a bound flavin semiquinone that appeared to interact magnetically with the ferric heme iron. NO production was inhibited by carbon monoxide, implying a role for the heme groups in catalysis.

Topics: Amino Acid Oxidoreductases; Animals; Brain; Cell Line; Cytochrome P-450 Enzyme System; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Hemeproteins; Humans; Kidney; Kinetics; Macrophages; Mice; Nitric Oxide Synthase; Rats; Spectrophotometry; Transfection

Reduction of turnip ferricytochrome f by flavin semiquinones and oxidation of this ferrocytochrome f by French bean cupriplastocyanin are studied by laser flash photolysis over a wide range of ionic strengths. Second-order rate constants (+/- 15%) at extreme values of ionic strength, all at pH 7.0 and 22 degrees C, are as follows: with FMN semiquinone at 1.00 and 0.0040 M, 5.0 x 10(7) and 3.9 x 10(8) M-1 s-1; with riboflavin semiquinone at 1.00 and 0.0040 m, 1.7 x 10(8) and 1.9 x 10(8) M-1 s-1; with lumiflavin semiquinone at 1.00 and 0.0045 M, 1.8 x 10(8) and 4.5 x 10(8) M-1 s-1; with cupriplastocyanin at 1.00 and 0.100 M, 1.4 x 10(6) and 2.0 x 10(8) M-1 s-1. These reactions of cytochrome f are governed by the local positive charge of the interaction domain (the exposed heme edge), not by the overall negative charge of the protein. Lumiflavin semiquinone behaves as if it carried a small negative charge, probably because partial localization of the odd electron gives this electroneutral molecule some polarity; local charge seems to be more important than overall charge even for relatively small redox agents. The dependence of the rate constants on ionic strength was fitted to the equation of Watkins; this model recognizes the importance of local charges of the domains through which redox partners interact. There is kinetic evidence that a noncovalent complex between cytochrome f and plastocyanin exists at low ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cytochromes; Cytochromes f; Electrochemistry; Electrons; Fabaceae; Flavin-Adenine Dinucleotide; Kinetics; Osmolar Concentration; Oxidation-Reduction; Plants, Medicinal; Plastocyanin; Spectrum Analysis; Vegetables

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39-45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J. M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491-503]. In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs-pyruvate complex is actually much more stable than the Fo-pyruvate and Fh-pyruvate complexes. The inhibition produced by pyruvate has been characterized under steady-state conditions using both ferricytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead-end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.

Topics: Cytochrome c Group; Electron Transport; Ferricyanides; Flavin-Adenine Dinucleotide; Flavins; Kinetics; L-Lactate Dehydrogenase; L-Lactate Dehydrogenase (Cytochrome); Lactates; Mathematics; Pyruvates; Thermodynamics

Steady-state and laser flash photolysis techniques have been used to examine the photoreduction of yeast glutathione reductase by the one-electron reduction products of 5-deazariboflavin and the viologen analogue 1,1'-propylene-2,2'-bipyridyl. Steady-state photoreduction of the enzyme with the viologen generates the two-electron-reduced form, whereas photoreduction with deazaflavin generates the anion semiquinone. Flash photolysis indicates that the product of viologen radical reduction is also a semiquinone, suggesting that this species is rapidly further reduced by viologen in the steady-state experiment to form the EH2 enzyme. This reduction is apparently inhibited when deazaflavin is the photoreductant, perhaps due to complexation of the anion semiquinone with deazaflavin. Steady-state experiments demonstrate that complexation of the anion semiquinone with NADP+ also inhibits further reduction. Both one-electron reduction reactions of oxidized glutathione reductase proceed at close to diffusion-controlled rates (second-order rate constants = 10(8)-10(9) M-1 s-1), despite the relatively buried nature of the FAD cofactor. Addition of NADP+ and oxidized glutathione produced no effects on the kinetics of the initial entry of the electron into the enzyme. No kinetic evidence of intramolecular electron transfer involving the FAD and the protein disulfide was obtained during or subsequent to the initial one-electron reduction process. Thus, if this reaction occurs in the semiquinone, it must be quite rapid (k greater than 8000 s-1).

Topics: Chemical Phenomena; Chemistry; Diquat; Electrons; Flavin-Adenine Dinucleotide; Free Radicals; Glutathione; Glutathione Reductase; Kinetics; Lasers; NADP; Oxidation-Reduction; Photochemistry; Riboflavin; Time Factors; Viologens; Yeasts

Spinach plastocyanin binds to both electrically neutral and positively charged lipid bilayer vesicles, whereas cytochrome c only binds electrostatically to negatively charged vesicles. Laser flash photolysis using lumiflavin semiquinone as a reductant demonstrates that the reactivity of plastocyanin is increased as much as 6-fold when it is membrane bound whereas the rate constant for cytochrome c reduction is decreased by approximately a factor of 3. Membrane-bound plastocyanin reduction occurs via a two-step mechanism, probably involving prior association of lumiflavin semiquinone with the bilayer. In contrast, cytochrome c reduction in the membrane-bound state follows simple second-order kinetics, implying that the redox site in the bound state is still accessible to lumiflavin semiquinone in solution, although the rate constant is decreased by approximately 3-fold. These results are interpreted as indicating that the bilayer-protein interaction with plastocyanin leads to a steric blockage of the electron-transfer site from the aqueous phase. Little or no hindrance of the redox site occurs with cytochrome c, suggesting a high degree of mobility of this protein on the bilayer surface. Although the increase in plastocyanin reactivity upon binding to the bilayer is quite interesting, its cause remains unclear and requires further study. The results illustrate the utility of laser flash photolysis as a probe of membrane-protein interactions.

Topics: Animals; Cytochrome c Group; Flavin-Adenine Dinucleotide; Horses; Kinetics; Lasers; Lipid Bilayers; Myocardium; Oxidation-Reduction; Phosphatidylcholines; Photolysis; Plant Proteins; Plants; Plastocyanin; Protein Binding

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast ferric and ferryl cytochrome c peroxidase and the cytochromes c of horse, tuna, and yeast (iso-2) have been studied. Complex formation decreases the rate constant for reduction of ferric peroxidase by 44%. On the basis of a computer model of the complex structure [Poulos, T.L., & Finzel, B.C. (1984) Pept. Protein Rev. 4, 115-171], this decrease cannot be accounted for by steric effects and suggests a decrease in the dynamic motions of the peroxidase at the peroxide access channel caused by complexation. The orientations of the three cytochromes within the complex are not equivalent. This is shown by differential decreases in the rate constants for reduction by neutral flavin semiquinones upon complexation, which are in the order tuna much greater than horse greater than yeast iso-2. Further support for differences in orientation is provided by the observation that, with the negatively charged reductant FMNH., the electrostatic environments near the horse and tuna cytochrome c electron-transfer sites within their respective complexes with peroxidase are of opposite sign. For the horse and tuna cytochrome c complexes, we have also observed nonlinear concentration dependencies of the reduction rate constants with FMNH.. This is interpreted in terms of dynamic motion at the protein-protein interface. We have directly measured the physiologically significant intra-complex one electron transfer rate constants from the three ferrous cytochromes c to the peroxide-oxidized species of the peroxidase. At low ionic strength these rate constants are 920, 730, and 150 s-1 for tuna, horse, and yeast cytochromes c, respectively. These results are also consistent with the contention that the orientations of the three cytochromes within the complex with CcP are not the same. The effect on the intracomplex electron-transfer rate constant of the peroxidase amino acid side chain(s) that is (are) oxidized by the reduction of peroxide was determined to be relatively small. Thus, the rate constant for reduction by horse cytochrome c of the peroxidase species in which only the heme iron atom is oxidized was decreased by only 38%, indicating that this oxidized side-chain group is not tightly coupled to the ferryl peroxidase heme iron. Finally, it was found that, in the absence of cytochrome c, neither of the ferryl peroxidase species could be rapidly reduced by flavin semiq

Topics: Animals; Cytochrome c Group; Cytochrome-c Peroxidase; Electron Transport; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Horses; Kinetics; Oxidation-Reduction; Peroxidases; Protein Conformation; Saccharomyces cerevisiae; Tuna

NADPH-adrenodoxin oxidoreductase was titrated with NADPH under anaerobic conditions. As the amount of added NADPH was increased to a ratio to the reductase of 1 : 1, a broad absorbance band from approximately 500 to 900 nm, which is attributed to a charge transfer complex, increased and then sharply decreased after the 1 : 1 ratio was attained. Concomitant with the decrease in the charge transfer band, a peak at 575 nm with a shoulder at 635 nm increased, indicating the formation of a semiquinone. This showed clearly that a semiquinone was formed only when more than the stoichiometric amount of NADPH (It is meant by "the stoichiometric amount of NADPH" that the molar ratio of NADPH to adrenodoxin reductase is equal to one, that is, NADPH/FAD bound to the reductase = 1.) was added. The semiquinone band reached its maximum with an approximately 3-fold excess of NADPH over the reductase, and then gradually decreased. Concurrent with the decrease in absorbance of both the charge transfer complex and the semiquinone, the reaction mixture was bleached, indicating that a pale colored species was produced. 1H NMR studies suggested that the pale colored species was a complex of fully reduced adrenodoxin reductase and NADPH, and that the semiquinone also bound 1 mol of the pyridine nucleotide per mol of the reductase. These data suggest that the semiquinone state of the reductase is observable only when a complex between NADPH and the enzyme in the flavin semiquinone is formed.

Topics: Anaerobiosis; Dithionite; Electron Transport; Ferredoxin-NADP Reductase; Flavin-Adenine Dinucleotide; Magnetic Resonance Spectroscopy; NADH, NADPH Oxidoreductases; NADP; Oxidation-Reduction; Spectrophotometry

We have measured the ionic strength dependence of the rate constants for electron transfer from the semiquinone of Clostridium pasteurianum flavodoxin to 12 c-type cytochromes and several inorganic oxidants using stopped-flow methodology. The experimental data were fit quite well by an electrostatic model that represents the interaction domains as parallel disks with a point charge equal to the charge within this region of the protein. The analysis provides an evaluation of the electrostatic interaction energy and the rate constant at infinite ionic strength (k affinity). The electrostatic charge on the oxidant within the interaction site can be obtained from the electrostatic energy, and for most of those reactants for which structures are available, the results are in good agreement with expectation. The k affinity values were found to correlate with redox potential differences, as expected from the theory of adiabatic (or nonadiabatic) outer-sphere electron-transfer reactions. Deviations from the theoretical curves are interpreted in terms of the influence of surface topology on reaction rate constants. In general, we find that electrostatic effects, steric influences, and redox potential all exert a much larger effect on reaction rate constants for the flavodoxin-cytochrome system than has been previously observed for free flavin-cytochrome interactions. The implications of this for determining biological specificity are discussed.

Topics: Clostridium; Cytochrome c Group; Electrochemistry; Electron Transport; Flavin-Adenine Dinucleotide; Flavodoxin; Flavoproteins; Kinetics; Osmolar Concentration; Oxidation-Reduction; Thermodynamics

The reduction of D-amino acid oxidase (DAAO) by hydrated electrons (eaq-) has been studied in the absence and presence of benzoate by pulse radiolysis. The eaq-did not reduce the flavin moiety in DAAO and reacted with the amino acid residues in the protein. In the presence of benzoate, eaq- first reacted with benzoate to yield benzoate anion radical. Subsequently, the benzoate anion radical transferred an electron to the complex of DAAO-benzoate to form the red semiquinone of the enzyme with a second-order rate constant of 1.2 X 10(9) M-1 s-1 at pH 8.3. After the first phase of the reduction, conversion of the red semiquinone to the blue semiquinone was observed in the presence of high concentration of benzoate. This process obeyed first-order kinetics, and the rate increased with an increase of the concentration of benzoate. In addition, the rate was found to be identical with that of the formation of the complex between benzoate and the red semiquinone of DAAO as measured by a stopped-flow method. This suggests that bound benzoate dissociates after the reduction of the benzoate-DAAO complex by benzoate anion radical and that free benzoate subsequently recombines with the red semiquinone of the enzyme to form the blue semiquinone.

Topics: Animals; Benzoquinones; D-Amino-Acid Oxidase; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Kidney; Kinetics; Quinones; Swine

Resonance Raman spectra are reported for the semiquinone of N5-methyl derivatives of FMN (flavin mononucleotide) in H2O and 2H2O, 8-chloro FMN and FAD (flavin adenine dinucleotide) with 647.1 nm excitation, in the first pi-pi absorption band, using KI to quench fluorescence. The spectral pattern is similar to that of oxidized flavin, in its first absorption band, but with appreciable shifts, up to approx. 50 cm-1, in corresponding frequencies. There are also significant shifts with respect to the previously reported resonance Raman spectrum of flavodoxin semiquinone, reflecting the substitution of CH3 for H at N5. The N5-methyl FAD semiquinone spectrum is also reported for 514.5 nm excitation, in resonance with the second pi-pi transition. The intensity pattern is quite different, the spectrum being dominated by a band at 1611 cm-1, assigned to a mode localized primarily on the central pyrazine ring.

Topics: Carrier Proteins; Drug Stability; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Membrane Transport Proteins; Methylation; Riboflavin; Spectrophotometry; Spectrum Analysis, Raman; Structure-Activity Relationship

The relaxation behavior of the EPR signals of MoV, FAD semiquinone, and the reduced Fe/S I center was measured in the presence and absence of other paramagnetic centers in milk xanthine oxidase. Specific pairs of prosthetic groups were rendered paramagnetic by poising the native enzyme or its desulfo glycol inhibited derivative at appropriate potentials and pH values. Magnetic interactions were found between the following species: Mo--Fe/S I (100-fold increase in microwave power required to saturate the MoV EPR signal at 103 K when Fe/S I is reduced as opposed to oxidized), FAD--Fe/S I and FAD--Fe/S II (70-fold increase in power required to saturate the FADH.EPR signal at 173 K when either Fe/S center is reduced), and Fe/S I--Fe/S II (2.5-fold increase in power to saturate the reduced Fe/S I EPR signal at 20 K when Fe/S II is reduced). The Mo--Fe/S I interaction was also detected as a reduced Fe/S I induced splitting of the MoV EPR spectrum at 30 K. No splittings of the FADH. or Fe/S center spectra were detected. No magnetic interactions were found between FAD and Mo or between Mo and Fe/S II. These results, together with those of Coffman & Buettner [Coffman, R. E., & Buettner, G. R. (1979) J. Phys. Chem. 83, 2392-2400], were used to estimate the following approximate distances between the electron carrying prosthetic groups of milk xamthine oxidase: Mo--Fe/S I, 11 +/- 3 A; Fe/S I-Fe/S II, 15 +/- 4 A; FAD-Fe/S I, 16 +/- 4 A; FAD-Fe/S II, 16 +/- 4 A. A model for the arrangement of these groups within the xanthine oxidase molecule is suggested.

Topics: Animals; Cattle; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Iron; Magnetics; Milk; Molybdenum; Oxidation-Reduction; Sulfur; Xanthine Oxidase

Topics: Animals; Cattle; Diphosphates; Electrochemistry; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Iron; Milk; Molybdenum; Oxidation-Reduction; Sulfur; Xanthine Oxidase

Topics: Animals; Cattle; Chemical Phenomena; Chemistry; Flavin-Adenine Dinucleotide; Hydrogen-Ion Concentration; Iron; Milk; Molybdenum; Oxidation-Reduction; Sulfur; Uric Acid; Xanthine Oxidase

Methanol oxidase from Hansenula polymorpha contains five "red" flavin semiquinones and two oxidized flavins per octamer. Addition of substrate results in the reduction of the two oxidized flavins but does not affect the flavin semiquinones. Enhanced water proton relaxation rates indicate that the unpaired electron of the flavin semiquinones is accessible to the solvent and this accessibility is significantly decreased upon binding of the suicide inhibitor cyclopropanol. In the native enzyme, the semiquinones are not oxidizable by air. All flavins were resolved from the enzyme, and holoenzyme was reconstituted by addition of oxidized flavin. The reconstituted enzyme was catalytically active. The specific activity was 50% that of the original enzyme. It was concluded that the semiquinone is not required for the oxidation of methanol, although it may be present at an otherwise intact site.

Topics: Alcohol Oxidoreductases; Apoenzymes; Ascomycota; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Macromolecular Substances; Pichia; Spectrum Analysis

Topics: Animals; Catalysis; Cattle; Chromatography, Gel; Electron Spin Resonance Spectroscopy; Flavin-Adenine Dinucleotide; Lacticaseibacillus casei; Mitochondria; Monoamine Oxidase