Amino Acid Oxidase Research Articles

Online oxygen kinetic isotope effects using membrane inlet mass spectrometry can differentiate between oxidases for mechanistic studies and calculation of their contributions to oxygen consumption in whole tissues.

The reduction chemistry of molecular oxygen underpins the energy metabolism of multicellular organisms, liberating free energy needed to catalyze a plethora of enzymatic reactions. Measuring the isotope signatures of (16)O and (18)O during O2 reduction can provide insights into both kinetic and equilibrium isotope effects. However, current methods to measure O2 isotope signatures are time-consuming and disruptive. This paper describes the application of membrane inlet mass spectrometry to determine the oxygen isotope discrimination of a range of O2-consuming reactions, providing a rapid and convenient method for determining these values. A survey of oxygenase and oxidase reactions provides new insights into previously uncharacterized amino acid oxidase enzymes. Liquid and gas phase measurements show the ease of assays using this approach for purified enzymes, biological extracts and intact tissues.

Dissecting Physical and Biochemical Factors of Catalytic Effectiveness in Immobilized D‐Amino Acid Oxidase by Real‐Time Sensing of O2 Availability Inside Porous Carriers

AbstractD‐Amino acid oxidase (DAAO) presents a paragon for effective use of biocatalytic O2‐dependent transformations in fine‐chemical and pharmaceutical synthesis at the industrial scale. Solid‐supported DAAO immobilizates are applied to continuous processing, but their activity and stability are often inadequate. Targeted immobilization development is restricted by insufficient knowledge of physical and biochemical factors governing the performance of heterogeneous DAAO catalysts. We have applied real‐time optical sensing of the O2 availability in luminescence‐labeled porous Sepabeads and ReliSorb carriers to quantify diffusional restrictions in DAAO immobilizates differing in the mode of enzyme attachment to the solid surface. We show that noncovalent oriented immobilization of DAAO (from Trigonopsis variabilis) resulted in high retention of the original enzyme activity (≥60 %), whereas covalent multipoint fixation caused massive (up to 90 %) activity loss. Depletion of O2 inside the solid immobilizates became limiting for enzyme catalytic effectiveness at activity loadings as low as 5 units gcarrier−1. Slow pore diffusion was principally responsible for the observed large mass‐transfer resistance, and this provides a main starting point for process intensification.

Tailoring D‐Amino Acid Oxidase from the Pig Kidney to R‐Stereoselective Amine Oxidase and its Use in the Deracemization of α‐Methylbenzylamine

The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, R-stereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney D-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate α-methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure-activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.

Tailoring D‐Amino Acid Oxidase from the Pig Kidney to R‐Stereoselective Amine Oxidase and its Use in the Deracemization of α‐Methylbenzylamine

AbstractThe deracemization of racemic amines to yield enantioenriched amines using S‐stereoselective amine oxidases (AOx) has recently been attracting attention. However, R‐stereoselective AOx that are suitable for deracemization have not yet been identified. An R‐stereoselective AOx was now evolved from porcine kidney D‐amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate α‐methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure–activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.

Molecular characterization of Rhodnius prolixus' embryonic cuticle

The embryonic cuticle (EC) of Rhodnius prolixus envelopes the entire body of the embryo during hatching and provides physical protection, allowing the embryo to pass through a narrow chorionic border. Most of the knowledge about the EC of insects is derived from studies on ultrastructure and secretion processes during embryonic development, and little is known about the molecular composition of this structure. We performed a comprehensive molecular characterization of the major components extracted from the EC of R. prolixus, and we discuss the role of the different molecules that were identified during the eclosion process. The results showed that, similar to the post-embryonic cuticles of insects, the EC of R. prolixus is primarily composed of carbohydrates (57%), lipids (19%), and proteins (8%). Considering only the carbohydrates, chitin is by far the major component (approximately 70%), and it is found primarily along the body of the EC. It is scarce or absent in its prolongations, which are composed of glycosaminoglycans. In addition to chitin, we also identified amino (15%), neutral (12%) and acidic (3%) carbohydrates in the EC of R. prolixus. In addition carbohydrates, we also identified neutral lipids (64.12%) and phospholipids (35.88%). Proteomic analysis detected 68 proteins (55 were identified and 13 are hypothetical proteins) using the sequences in the R. prolixus genome (http://www.vectorbase.org). Among these proteins, 8 out of 15 are associated with cuticle metabolism. These proteins are unequivocally cuticle proteins, and they have been described in other insects. Approximately 35% of the total proteins identified were classified as having a structural function. Chitin-binding protein, amino peptidase, amino acid oxidase, oxidoreductase, catalase and peroxidase are all proteins associated with cuticle metabolism. Proteins known to be cuticle constituents may be related to the function of the EC in assisting the insect during eclosion. To our knowledge, this is the first study to describe the global molecular composition of an EC in insects.

Theoretical analyses of the fluorescence lifetimes of thed-amino acid oxidase–benzoate complex dimer from porcine kidney: molecular dynamics simulation and photoinduced electron transfer

The mechanism of photoinduced electron transfer from benzoate and aromatic amino acids to the excited isoalloxazine in thed-amino acid oxidase–benzoate complex dimer was studied using molecular dynamics simulation and an electron transfer theory.

Non-equivalent conformations ofd-amino acid oxidase dimer from porcine kidney between the two subunits. Molecular dynamics simulation and photoinduced electron transfer

The structural difference between two subunits of D-amino acid oxidase dimer from porcine kidney was studied by molecular dynamics simulation (MDS) and rate of photoinduced electron transfer (ET) from aromatic amino acids as tyrosines (Tyr) and tryptophanes (Trp) to the excited isoalloxazine (Iso*). The donor-acceptor distances (Rc) between isoalloxazine (Iso) and the donors were shortest in Tyr224 (0.74 nm) in Sub A at 10 °C (Sub A10), in Tyr224 (0.79 nm) in Sub B at 10 °C (Sub B10), in Tyr228 (0.85 nm) in Sub A at 30 °C (Sub A30), and in Tyr224 (0.72 nm) in Sub B at 30 °C (Sub B30). The Rcs were mostly shorter in the dimer than those in the monomer. Hydrogen bonding (H-bond) pairs between Iso and surrounding amino acids varied with the subunit and temperature. O2 of the Iso ring formed an H-bond exclusively with Thr317OG1 (side chain) in both Sub A10 and Sub A30, while it formed with Gly315N (peptide), Leu316N and Thr317N in Sub B10 and Sub B30. N3H of Iso formed an H-bond with Leu51O (peptide) in Sub A10 and Sub A30, but not in Sub B10 and Sub B30. Electron affinity of Iso* was appreciably lower in Sub A10 compared to Sub B10, while it was opposite at 30 °C. ET rate to Iso* was fastest from Tyr224 in Sub A10, while it was fastest from Tyr314 in Sub B10. The ET rate was fastest from Tyr314 in Sub A30, while it was fastest from Tyr224 in Sub B30. The greater ET rates in the dimer as compared to those in the monomer were elucidated with shorter Rc in the dimer as compared to the monomer. The static dielectric constants inside the subunits and the static dielectric constant between Iso and Tyr224 or Tyr228 were not different appreciably. A few water molecules and sometimes an amino acid were located between Iso and Tyr224, which may be the reason why the dielectric constant of the entire subunits did not differ from that between Iso and Tyr224.

The degradation (by distinct pathways) of human d‐amino acid oxidase and its interacting partner pLG72 – two key proteins in d‐serine catabolism in the brain

Human D-amino acid oxidase (EC 1.4.3.3; hDAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. hDAAO has been proposed to play (with serine racemase; EC 5.1.1.18) an essential role in the catabolism of D-serine, an 'atypical' key signalling molecule that acts as allosteric activator of the N-methyl-D-aspartate-type glutamate receptor (NMDAr). hDAAO and its interacting partner pLG72 have been related to schizophrenia, a highly disabling psychiatric disorder in which a dysfunction of NMDA-mediated neurotransmission is widely assumed to occur. We previously demonstrated that the D-serine cellular concentration depends on hDAAO and pLG72 expression levels and that newly-synthesized hDAAO interacts with its modulator in the cytosol, being progressively destabilized and inactivated. To obtain insight into the mechanisms regulating cellular D-serine levels, we investigated the degradation pathways of hDAAO and pLG72 in U87 glioblastoma cells stably expressing enhanced yellow fluorescent protein-hDAAO (peroxisomal), hDAAO-enhanced yellow fluorescent protein (cytosolic) or pLG72-enhanced cyan fluorescent protein (mitochondrial) proteins. hDAAO is a long-lived protein: the peroxisomal fraction of this flavoprotein is degraded via the lysosomal/endosomal pathway (and blocking this pathway increases the cellular hDAAO activity and decreases D-serine levels), whereas the cytosolic portion is ubiquitinated and targeted to the proteasome. By contrast, pLG72 shows a rapid turnover (t(1/2) ≈ 25-40 min) and is degraded via the proteasome system, albeit not ubiquitinated. Overexpression of pLG72 increases the turnover of hDAAO, in turn playing a protective role against excessive D-serine depletion.

D -Amino Acid Oxidase–Nanoparticle System: A Potential Novel Approach for Cancer Enzymatic Therapy

The authors propose a new magnetic nanoparticle-enzyme system for cancer therapy capable of targeting the enzyme and consequently decreasing the adverse effects, meanwhile improving the patient's life quality. The authors have functionalized Fe3O4 nanoparticles with 3-amino-propyltriethoxysilane (APTES) and conjugated it to yeast D-amino acid oxidase (DAAO) by coupling this with glutaraldehyde. The authors have tested the Fe3O4-APTES-DAAO system on three tumor cell lines. Exposed cells show, at the electron microscope level, nanoparticles on the surface of the plasma membrane and inside endocytic vesicles. Fe3O4-APTES-DAAO caused a substantial decrease of cell viability greatly augmented when D-alanine, a DAAO substrate, was added. Fe3O4-APTES-DAAO was demonstrated to be more effective than free DAAO, confirming the validity of the system in cancer therapy.

Pathogenic effects of amyotrophic lateral sclerosis-linked mutation in D-amino acid oxidase are mediated by D-serine

Amyotrophic lateral sclerosis is a neuromuscular disease characterized by selective loss of motor neurons leading to fatal paralysis. We previously reported a coding mutation in D–amino acid oxidase (R199W DAO) associated with familial amyotrophic lateral sclerosis. DAO metabolizes D-serine, a co-agonist at the N-methyl-D-aspartic acid receptor. We investigated the mechanisms mediating the pathogenic effects of R199W DAO on motor neuron survival and showed that expression of glial R199W DAO is sufficient to induce apoptosis in cocultured motor neurons and this is sensitive to 5,7-dichloro-4-hydroxyquinoline-2-carboxylic acid, an N-methyl-d-aspartic acid receptor antagonist selective for the D-serine/glycine site. R199W DAO activates protein aggregation and autophagy, which is also sensitive to this antagonist. Using immunocytochemistry, we showed that D-serine and DAO were abundant in spinal cord motor neurons and depleted in amyotrophic lateral sclerosis. In summary, the toxic effects of R199W DAO on motor neurons can be mediated directly by expression in motor neurons or by astrocytes in coculture, R199W DAO promotes autophagy and its pathogenic effects are at least in part mediated via the N-methyl-d-aspartic acid receptor.

L-Amino acid oxidase as biocatalyst: a dream too far?

L-amino acid oxidase (LAAO) is a flavoenzyme containing non-covalently bound flavin adenine dinucleotide, which catalyzes the stereospecific oxidative deamination of l-amino acids to α-keto acids and also produces ammonia and hydrogen peroxide via an imino acid intermediate. LAAOs purified from snake venoms are the best-studied members of this family of enzymes, although a number of LAAOs from bacterial and fungal sources have been also reported. From a biochemical point of view, LAAOs from different sources are distinguished by molecular mass, substrate specificity, post-translational modifications and regulation. In analogy to the well-known biotechnological applications of d-amino acid oxidase, important results are expected from the availability of suitable LAAOs; however, these expectations have not been fulfilled yet because none of the "true" LAAOs has successfully been expressed as a recombinant protein in prokaryotic hosts, such as Escherichia coli. In enzyme biotechnology, recombinant production of a protein is mandatory both for the production of large amounts of the catalyst and to improve its biochemical properties by protein engineering. As an alternative, flavoenzymes active on specific l-amino acids have been identified, e.g., l-aspartate oxidase, l-lysine oxidase, l-phenylalanine oxidase, etc. According to presently available information, amino acid oxidases with "narrow" or "strict" substrate specificity represent as good candidates to obtain an enzyme more suitable for biotechnological applications by enlarging their substrate specificity by means of protein engineering.

Retinal NMDA receptor function and expression are altered in a mouse lackingd-amino acid oxidase

D-serine is present in the vertebrate retina and serves as a coagonist for the N-methyl-D-aspartate (NMDA) receptors of ganglion cells. Although the enzyme D-amino acid oxidase (DAO) has been implicated as a pathway for d-serine degradation, its role in the retina has not been established. In this study, we investigated the role of DAO in regulating D-serine levels using a mutant mouse line deficient in DAO (ddY/DAO(-)) and compared these results with their wild-type counterparts (ddY/DAO(+)). Our results show that DAO is functionally present in the mouse retina and normally serves to reduce the background levels of D-serine. The enzymatic activity of DAO was restricted to the inner plexiform layer as determined by histochemical analysis. Using capillary electrophoresis, we showed that mutant mice had much higher levels of D-serine. Whole cell recordings from identified retinal ganglion cells demonstrated that DAO-deficient animals had light-evoked synaptic activity strongly biased toward a high NMDA-to-AMPA receptor ratio. In contrast, recordings from wild-type ganglion cells showed a more balanced ratio between the two receptor subclasses. Immunostaining for AMPA and NMDA receptors was carried out to compare the two receptor ratios by quantitative immunofluorescence. These studies revealed that the mutant mouse had a significantly higher representation of NMDA receptors compared with the wild-type controls. We conclude that 1) DAO is an important regulatory enzyme and normally functions to reduce D-serine levels in the retina, and 2) D-serine levels play a role in the expression of NMDA receptors and the NMDA-to-AMPA receptor ratio.

D-Amino acid oxidase and presence of d-proline in Xenopus laevis

We purified D-amino acid oxidase (EC 1.4.3.3, DAO) from Xenopus laevis tadpoles. The optimal temperature and pH for enzyme activity were 35-40 °C and 8.3-9.0, respectively, depending on the substrate amino acids available to the enzyme; the highest activity was observed with D-proline followed by D-phenylalanine. Activity was significantly inhibited by p-hydroxymercuribenzoate, but only moderately by p-chloromercuribenzoate or benzoate. Enzyme activity was increased until the final tadpole stage, but was reduced to one-third in the adult and was localized primarily in the kidney. The tadpoles contained high concentrations of D-proline close to the final developmental stage and nearly no D-amino acids were detected in the adult frog, indicating that D-amino acid oxidase functions in metamorphosis.

Physiological function of hydrogen sulfide and beyond

The relatively high concentrations of endogenous sulfide in the mammalian brain were measured in 1989, suggesting that hydrogen sulfide (H2S) might have a physiological function. In 1996 we demonstrated that cystathionine β-synthase (CBS) is a H2S producing enzyme in the brain and that H2S facilitates the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory, by enhancing the activity of N-methyl d -aspartate (NMDA) receptors. The following year we demonstrated that another H2S producing enzyme, cystathionine γ-lyase (CSE) is found in tissues including vasculature and that H2S relaxes them. Based on these observations we proposed that H2S is a neuromodulator and a smooth muscle relaxant. In addition to the function as a signaling molecule, we and others found a cytoprotective effect of this molecule; H2S protects neurons from oxidative stress. This finding led to the identification of the protection of various organs including the heart, pancreas, retina, and the kidney against ischemia–reperfusion injury. From our finding that the brains of CBS knockout mice was still able to produce H2S, we found another pathway; 3-mercaptopyruvate sulfur transferase (3MST) along with cysteine aminotransferase (CAT). 3MST produces H2S in the presence of thioredoxin or dihydrolipoic acid (DHLA). We recently found a novel pathway to produce H2S from d -cysteine, a negative control. d -Amino acid oxidase (DAO) metabolizes d -cysteine to an achiral α–keto acid, 3-mercaptopyruvate (3MP), which is further metabolized to H2S by 3MST. This pathway is mainly localized in the cerebellum and the kidney. The production of H2S from d -cysteine is 80 times more efficient than that from l -cysteine in the kidney, and the administration of d -cysteine to mice ameliorates renal ischemia–reperfusion injury more effectively than l -cysteine. These results show a therapeutic potential of d -cysteine to the renal diseases and even to the kidney transplantation. Our additional contribution to this field is the discovery of H2S-derived polysulfides, which exist in the brain and activate transient receptor potential ankyrin-1 (TRPA1) channels 300 times more potently than H2S. TRPA1 channels mediate the sensory transduction and respond to a variety of stimuli, including cold temperature, pungent compounds and environmental irritants, but its endogenous ligand has not been identified. The sulfane sulfur of polysulfides is reactive electrophile and readily transferred to a nucleophilic protein thiolate, to generate the protein persulfide (sulfhydration). The sulfhydration activity of polysulfides is much greater than H2S. The production of H2S and the physiological function of H2S and polysulfides will be discussed.

Structural, Kinetic, and Pharmacodynamic Mechanisms of d-Amino Acid Oxidase Inhibition by Small Molecules

We characterized the mechanism and pharmacodynamics of five structurally distinct inhibitors of d-amino acid oxidase. All inhibitors bound the oxidized form of human enzyme with affinity slightly higher than that of benzoate (Kd ≈ 2-4 μM). Stopped-flow experiments showed that pyrrole-based inhibitors possessed high affinity (Kd ≈ 100-200 nM) and slow release kinetics (k < 0.01 s(-1)) in the presence of substrate, while inhibitors with pendent aromatic groups altered conformations of the active site lid, as evidenced by X-ray crystallography, and showed slower kinetics of association. Rigid bioisosteres of benzoic acid induced a closed-lid conformation, had slower release in the presence of substrate, and were more potent than benzoate. Steady-state d-serine concentrations were described in a PK/PD model, and competition for d-serine sites on NMDA receptors was demonstrated in vivo. DAAO inhibition increased the spatiotemporal influence of glial-derived d-serine, suggesting localized effects on neuronal circuits where DAAO can exert a neuromodulatory role.

4-Hydroxypyridazin-3(2H)-one Derivatives as Novel d-Amino Acid Oxidase Inhibitors

D-Amino acid oxidase (DAAO) catalyzes the oxidation of d-amino acids including d-serine, a coagonist of the N-methyl-d-aspartate receptor. We identified a series of 4-hydroxypyridazin-3(2H)-one derivatives as novel DAAO inhibitors with high potency and substantial cell permeability using fragment-based drug design. Comparisons of complex structures deposited in the Protein Data Bank as well as those determined with in-house fragment hits revealed that a hydrophobic subpocket was formed perpendicular to the flavin ring by flipping Tyr224 in a ligand-dependent manner. We investigated the ability of the initial fragment hit, 3-hydroxy-pyridine-2(1H)-one, to fill this subpocket with the aid of complex structure information. 3-Hydroxy-5-(2-phenylethyl)pyridine-2(1H)-one exhibited the predicted binding mode and demonstrated high inhibitory activity for human DAAO in enzyme- and cell-based assays. We further designed and synthesized 4-hydroxypyridazin-3(2H)-one derivatives, which are equivalent to the 3-hydroxy-pyridine-2(1H)-one series but lack cell toxicity. 6-[2-(3,5-Difluorophenyl)ethyl]-4-hydroxypyridazin-3(2H)-one was found to be effective against MK-801-induced cognitive deficit in the Y-maze.

Selective tryptophan determination using tryptophan oxidases involved in bis-indole antibiotic biosynthesis

A novel tryptophan assay was developed using tryptophan oxidases. Although many l-amino acid oxidases (LAAOs) have been reported to catalyze tryptophan oxidation, most of them have broad substrate specificity and oxidize multiple amino acids besides tryptophan. To obtain a tryptophan-specific LAAO, we focused on bis-indole antibiotic biosynthesis, a bacterial secondary metabolic pathway. A putative LAAO from Streptomyces sp. TP-A0274, StaO involved in staurosporine biosynthesis, was heterologously expressed, biochemically characterized, and shown to serve as a selective tryptophan oxidase for the first time. In addition, another LAAO, VioA involved in violacein biosynthesis in Chromobacterium violaceum, was characterized for comparison with StaO. Interestingly, StaO and VioA share similar properties, namely narrow substrate specificity and high affinity for l-tryptophan, despite the phylogenetic distance between these enzymes. Owing to these features, uncommon among known LAAOs, StaO and VioA assays can be used for selective and accurate quantification of l-tryptophan via a coupled colorimetric reaction. Indeed, StaO and VioA assays provided tryptophan concentrations in human plasma as accurately as those obtained by high-performance liquid chromatography. Therefore, these enzymes were clearly shown to offer an effective method for determining tryptophan in biological samples rapidly, inexpensively, and accurately. The results shown here also suggest the possibility of metabolism-oriented screening as a strategy to obtain enzymes highly selective for individual biomolecules.

Identification of Novel d-Amino Acid Oxidase Inhibitors by in Silico Screening and Their Functional Characterization in Vitro

D-amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including D-serine and D-alanine, which are potential coagonists of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders such as schizophrenia. Hence, a DAO inhibitor that augments the brain levels of D-serine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure-function relationships of DAO.

Characterization and application of a l-specific amino acid oxidase from Rhodococcus sp. AIU LAB-3

An L-specific amino acid oxidase (L-AAO) suitable for assay of N-acyl-L-amino acid amidohydrolase (L-aminoacylase) activity was purified from Rhodococcus sp. AIU LAB-3. The enzyme exhibited broad substrate specificity and catalyzed an oxidative deamination of the a-amino group of L-amino acids. The optimal enzyme activities for L-amino acids tested were observed in the pH range from 6.0 to 8.5, and more than 80% of the maximum activity was obtained at pH 7.5. The enzyme was stable in the pH range from 7.0 to 8.5, and the apparent Km values for those L-amino acids were small. We, therefore, developed a new enzymatic method for assay of L-aminoacylase activity using the L-AAO at pH 7.5. The new enzymatic method had advantages that the L-aminoacylase reaction was spectrophotometrically followed by measuring absorbance at 555 nm. The L-aminoacylase activity was assayed within 10 min using a small reaction volume. Thus, the new enzymatic method was simple and sensitive compared to the ninhydrin method.

The mechanism of photoinduced electron transfer in the d-amino acid oxidase–benzoate complex from pig kidney: Electron transfer in the inverted region

Abstract d -Amino acid oxidase (DAAO) from pig kidney binds flavin adenine dinucleotide (FAD) as a cofactor. The DAAO–benzoate (Bz) complex (DAOB) is remarkably more stable than DAAO. It was previously reported that the fluorescence lifetime of FAD in DAAO is reduced by 66% upon Bz binding. The mechanism of photoinduced electron transfer (ET) in the DAOB monomer from Bz to the excited isoalloxazine (Iso*) was studied by molecular dynamics simulation (MDS) and by Kakitani and Mataga electron transfer theory (KM theory), and compared with that in DAAO. The ET parameters contained in KM theory were determined to obtain the fluorescence lifetime of DAOB (60 ps). The ET mechanism in DAOB is different from other flavoproteins, where neutral tryptophans and tyrosines are the ET donors, since the anionic Bz acts as the anionic ET donor in DAOB. Moreover, ET takes place in the inverted region. Charge transfer interaction (CT) was studied by semi-empirical molecular orbital (MO) methods. The dipole moment in the Iso*–Bz system was lower than that in the isoalloxazine (Iso)–Bz system, which was ascribed to CT from the negatively charged Bz to Iso*. The mean interaction energies between Iso and Bz over 100 snapshots were −16.8 and −8.1 kcal/mol in the excited and the ground states, respectively, with mean charge densities for the excited and the ground states of −0.354 and −0.013 at Iso and −0.646 and −0.987 at Bz, respectively. The ionization potential of Bz was evaluated as 7.3 eV by ET analysis, which was in excellent agreement with that obtained by MO calculations. The dielectric constant between Iso and the donors was 2.2 in DAOB, compared to 5.8 in DAAO. According to the MDS, several water molecules existed around Iso in DAAO that were all removed upon Bz binding resulting in a strongly non-polar environment near Iso in DAOB.