Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins.

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light-induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3-H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low-temperature Fourier transform infrared spectroscopy. Using specifically 15N-labeled FMN, [1,3-15N2]FMN, the N3-H stretch was identified at 2831 cm-1 for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3-H stretch showed temperature dependence, with a shift to lower frequencies at ≤200 K and to higher frequencies at ≥250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis phot1 and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neo1-LOV2 and neo1-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the ß-sheet region and/or the adjacent Jα helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neo1-LOV1 domain. The environment around the N3-H group was also investigated.

Interaction between NADH and electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii is a heterodimer containing two FAD cofactors. Isolated ETF contains only one FAD molecule, FAD-1, because the other, FAD-2, is lost during purification. FAD-2 is recovered by adding FAD to the isolated ETF. The two FAD molecules in holoETF were characterized using NADH. Spectrophotometric titration of isolated ETF with NADH showed a two-electron reduction of FAD-1 according to a monophasic profile indicating that FAD-1 receives electrons from NADH without involvement of FAD-2. When holoETF was titrated with NADH, FAD-2 was reduced to an anionic semiquinone and then was fully reduced before the reduction of FAD-1. The midpoint potential values at pH 7 were +81, -136 and -279 mV for the reduction of oxidized FAD-2 to semiquinone, semiquinone to the fully reduced FAD-2 and the two-electron reduction of FAD-1, respectively. Both FAD-1 and FAD-2 in holoETF were reduced by excess NADH very rapidly. The reduction of FAD-2 was slowed by replacement of FAD-1 with 8-cyano-FAD indicating that FAD-2 receives electrons from FAD-1 but not from NADH directly. The present results suggest that FAD-2 is the counterpart of the FAD in human ETF, which contains one FAD and one AMP.

Decomposition of the fluorescence spectra of two FAD molecules in electron-transferring flavoprotein from Megasphaera elsdenii.

Electron-transferring flavoprotein (ETF) from Megasphaera elsdenii contains two FAD molecules, FAD-1 and FAD-2. FAD-2 shows an unusual absorption spectrum with a 400-nm peak. In contrast, ETFs from other sources such as pig contain one FAD and one AMP with the FAD showing a typical flavin absorption spectrum with 380- and 440-nm peaks. It is presumed that FAD-2 is the counterpart of the FAD in other ETFs. In this study, the FAD-1 and FAD-2 fluorescence spectra were determined by titration of FAD-1-bound ETF with FAD using excitation-emission matrix (EEM) fluorescence spectroscopy. The EEM data were globally analysed, and the FAD fluorescence spectra were calculated from the principal components using their respective absorption spectra. The FAD-2 fluorescence spectrum was different from that of pig ETF, which is more intense and blue-shifted. AMP-free pig ETF in acidic solution, which has a comparable absorption spectrum to FAD-2, also had a similar fluorescence spectrum. This result suggests that FAD-2 in M. elsdenii ETF and the FAD in acidic AMP-free pig ETF share a common microenvironment. A review of published ETF fluorescence spectra led to the speculation that the majority of ETF molecules in solution are in the conformation depicted by the crystal structure.

FT-IR spectroscopic studies on the molecular mechanism for substrate specificity/activation of medium-chain acyl-CoA dehydrogenase.

The interactions of acyl-CoA with medium-chain acyl-CoA dehydrogenases (MCADs) reconstituted with artificial FADs-i.e. 8-CN-, 7,8-Cl(2)-, 8-Cl-, 8-OCH(3)- and 8-NH(2)-FAD-were investigated by UV-visible absorption and FT-IR measurements. Although 8-NH(2)-FAD-MCAD did not oxidize acyl-CoA the wavelength of the absorption maximum of the flavin was altered by acyl-CoAs binding. Thus, 8-NH(2)-FAD-MCAD is one of the attractive materials for investigation of enzyme-substrate (ES) interaction in ES complex (the complex of oxidized MCAD with acyl-CoA). FT-IR difference spectra between non-labelled and [1-(13)C]-labelled acyl-CoA free in solution and bound to oxidized 8-NH(2)-FAD-MCAD were obtained. The broad 1668-cm(-1) band of free octanoyl-CoA assigned to the C(1) = O stretching vibration appeared as a sharp signal at 1626 cm(-1) in the case of the complex. The downward shift indicates a large polarization of C(1) = O, and the sharpness suggests that the orientation of the C(1) = O in the active-site cavity is fairly limited. The hydrogen-bond enthalpy change responsible for the polarization on the transfer of the substrate from aqueous solution to the active site of MCAD was estimated to be approximately 15 kcal/mol. The 1626-cm(-1) band is noticeably weakened in the case of acyl-CoA with acyl chains longer than C12 which are poor substrates for MCAD, suggesting that C(1) = O is likely to exist in multiple orientations in the active-site cavity, whence the band becomes obscured. A band identical to that of bound C8-CoA was observed in the case of C4-CoA which is a poor substrate, indicating the strong hydrogen bond at C(1) = O.

Isomers in the excited state of electron-transferring flavoprotein from Megasphaera elsdenii: spectral resolution from the time-resolved fluorescence spectra.

Electron-transferring flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD's, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 degrees C and 25 degrees C. Wavelength-dependent fluorescence decays of the both ETF at 5 degrees C and 25 degrees C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 degrees C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 degrees C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 degrees C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 degrees C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.

Intramolecular and intermolecular perturbation on electronic state of FAD free in solution and bound to flavoproteins: FTIR spectroscopic study by using the C = O stretching vibrations as probes.

The intramolecular and intermolecular perturbation on the electronic state of FAD was investigated by FTIR spectroscopy by using the C=O stretching vibrations as probes in D(2)O solution. Natural and artificial FADs, i.e. 8-CN-, 8-Cl-, 8-H-, 8-OCH(3)-, and 8-NH(2)-FAD labelled by 2-(13)C, (18)O=C(2), or 4,10a-(13)C(2) were used for band assignments. The C(2)=O and C(4)=O stretching vibrations of oxidized FAD were shifted systematically by the substitution at the 8-position, i.e. the stronger the electron-donating ability (NH(2) > OCH(3) > CH(3) > H > Cl > CN) of the substituent, the lower the wavenumber region where both the C(2)=O and C(4)=O bands appear. In contrast, the C(4)=O band of anionic reduced FAD scarcely shifted. The 1,645-cm(-1) band containing C(2)=O stretching vibration shifted to 1,630 cm(-1) in the medium-chain acyl-CoA dehydrogenase (MCAD)-bound state, which can be explained by hydrogen bonds at C(2)=O of the flavin ring. The band was observed at 1,607 cm(-1) in the complex of MCAD with 3-thiaoctanoyl-CoA. The 23 cm(-1) shift was explained by the charge-transfer interaction between oxidized flavin and the anionic acyl-CoA. In the case of electron-transferring flavoprotein, two bands associated with the C(4)=O stretching vibration were obtained at 1,712 and 1,686 cm(-1), providing evidence for the multiple conformations of the protein.

Donor-acceptor distance-dependence of photoinduced electron-transfer rate in flavoproteins.

Ultrafast fluorescence quenching of flavin in flavodoxin from Megasphaera elsdenii was investigated by means of a fluorescence up-conversion method. Fluorescence lifetimes of flavodoxin from M. elsdenii were estimated to be tau(1) approximately 165 fs (0.97%) and tau(2) approximately 10 ps (0.03%). Correlation of photoinduced electron-transfer rates (k(ET)) with averaged distances (D(av)) between isoalloxazine and nearby tryptophan or tyrosine was examined and obtained an empirical equation of ln k(ET) vs D(av) by means of a nonlinear least-squares method using reported data together with flavodoxin from M. elsdenii. The values of D(av) were calculated from X-ray structures of the flavoproteins. The ln k(ET) was approximately linear at D(av) shorter than 7 A. The model free empirical equation was expressed as ln k(ET) = 29.7 + (-0.327 D(av) + 2.84 x 10(-5))/(0.698 - D(av)(2)). We also analyzed the observed values of ln k(ET) with Marcus theory, but could not obtain reasonable results. Our analysis suggests that the average distance, rather than the shortest (edge to edge) distance or interplanar angles between the aromatics rings, is the key factor in the process of the photoinduced electron transfer in these flavoproteins.

Identification of the C=O stretching vibrations of FMN and peptide backbone by 13C-labeling of the LOV2 domain of Adiantum phytochrome3.

Phototropin, a blue-light photoreceptor in plants, has two FMN-binding domains named LOV1 and LOV2. We previously observed temperature-dependent FTIR spectral changes in the C=O stretching region (amide-I vibrational region of the peptide backbone) for the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2), suggesting progressive structural changes in the protein moiety (Iwata, T., Nozaki, D., Tokutomi, S., Kagawa, T., Wada, M., and Kandori, H. (2003) Biochemistry 42, 8183-8191). Because FMN also possesses two C=O groups, in this article, we aimed at assigning C=O stretching vibrations of the FMN and protein by using 13C-labeling. We assigned the C(4)=O and C(2)=O stretching vibrations of FMN by using [4,10a-13C2] and [2-13C] FMNs, respectively, whereas C=O stretching vibrations of amide-I were assigned by using 13C-labeling of protein. We found that both C(4)=O and C(2)=O stretching vibrations shift to higher frequencies upon the formation of S390 at 77-295 K, suggesting that the hydrogen bonds of the C=O groups are weakened by adduct formation. Adduct formation presumably relocates the FMN chromophore apart from its hydrogen-bonding donors. Temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands. The hydrogen bond of the peptide backbone in the loop region is weakened upon S390 formation at low temperatures, while being strengthened at room temperature. The hydrogen bond of the peptide backbone in the alpha-helix is weakened regardless of temperature. On the other hand, structural perturbation of the beta-sheet is observed only at room temperature, where the hydrogen bond is strengthened. Light-signal transduction by phy3-LOV2 must be achieved by the progressive protein structural changes initiated by the adduct formation of the FMN.

Theoretical study on charge-transfer interaction between acyl-CoA dehydrogenase and 3-thiaacyl-CoA using density functional method.

Acyl-CoA dehydrogenase forms a complex with a substrate analog, 3-thiaacyl-CoA, exhibiting a charge-transfer (CT) band. The structure of a complex model of oxidized lumiflavin with deprotonated 3-thiabutanoate ethylthioester designed for the above CT complex was fully optimized by means of density functional theory (DFT), the spatial arrangement being similar to the corresponding X-ray structure reported previously. The electrostatic interaction between flavin and an anionic ligand, therefore, plays a major role in determination of the arrangement of the CT complex. When the excitation energies and oscillator strengths for the optimized structures of complex models including oxidized 8-substituted lumiflavins were calculated, the obtained wavelengths correlated well with observed values reported. Subsequently, we carried out DFT calculations for new complex models redesigned for complexes of oxidized 8-substituted FADs with an anionic ligand by introducing hydrogen bonds at the carbonyl group of the ligand with the 2'-hydroxyl group of the N10-ribityl of FAD and with the main-chain amide group of Glu376. The CT absorbing wavelengths of the new complex models exhibited better correlation with those observed previously. Consequently, comparison of substituent effects on the DFT calculations for the complex models will lead to a deeper understanding of the CT interaction and the effect of the hydrogen-bonding interaction on the CT framework.

Engineering the substrate specificity of porcine kidney D-amino acid oxidase by mutagenesis of the "active-site lid".

Comparison of the primary structures of pig kidney D-amino acid oxidase (DAO) and human brain D-aspartate oxidase (DDO) revealed a notable difference at I215-N225 of DAO and the corresponding region, R216-G220, of DDO. A DAO mutant, in which I215-N225 is substituted by R216-G220 of DDO, showed D-aspartate-oxidizing activity that wild-type DAO does not exhibit, together with a considerable decrease in activity toward D-alanine. These findings indicate that I215-N225 of DAO contributes profoundly to its substrate specificity. Based on these results and the crystal structure of DAO, we systematically mutated the E220-Y224 region within the short stretch in question and obtained five mutants (220D224G, 221D224G, 222D224G, 223D224G, and 224D), in each of which an aspartate residue is mutated to E220-Y224. All of the mutants exhibited decreased apparent K(m) values toward D-arginine, i.e., to one-seventh to one-half that of wild type DAO. The specificity constant, k(cat app)/K(m app), for D-arginine increased by one order of magnitude for the 221D224G or 222D224G mutant, whereas that for D-alanine or D-serine decreased to marginal or nil.

Three-dimensional structure of rat-liver acyl-CoA oxidase in complex with a fatty acid: insights into substrate-recognition and reactivity toward molecular oxygen.

The three-dimensional structure of rat-liver acyl-CoA oxidase-II (ACO-II) in a complex with a C12-fatty acid was solved by the molecular replacement method based on the uncomplexed ACO-II structure. The crystalline form of the complex was obtained by cocrystallization of ACO-II with dodecanoyl-CoA. The crystalline complex possessed, in the active-site crevice, only the fatty acid moiety that had been formed through hydrolysis of the thioester bond. The overall dimeric structure and the folding pattern of each subunit are essentially superimposable on those of uncomplexed ACO-II. The active site including the flavin ring of FAD, the crevice embracing the fatty acyl moiety, and adjacent amino acid side chains are superimposably conserved with the exception of Glu421, whose carboxylate group is tilted away to accommodate the fatty acid. One of the carboxyl oxygens of the bound fatty acid is hydrogen-bonded to the amide hydrogen of Glu421, the presumed catalytic base, and to the ribityl 2'-hydroxyl group of FAD. This hydrogen-bonding network correlates well with the substrate recognition/activation in acyl-CoA dehydrogenase. The binding mode of C12-fatty acid suggests that the active site does not close upon substrate binding, but remains spacious during the entire catalytic process, the oxygen accessibility in the oxidative half-reaction thereby being maintained.

Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis.

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis(Iseki et al., Nature, 2002, 415, 1047--1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACalpha and PACbeta). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD"(BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACalpha by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28--0.32, and the half-life was 34--44 s at 25 degrees C for the recombinant PACalpha F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACalpha F2 and the F2 domain of the PACalpha homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.

Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum.

Electron-transferring flavoprotein (ETF), its redox partner flavoproteins, i.e., D-lactate dehydrogenase and butyryl-CoA dehydrogenase, and another well-known flavoprotein, flavodoxin, were purified from the same starting cell paste of an anaerobic bacterium, Megasphaera elsdenii. The purified ETF contained one mol FAD/mol ETF as the sole non-protein component and bound almost one mol of additional FAD. This preparation is a better subject for investigations of M. elsdenii ETF than the previously isolated ETF, which contains varying amounts of FAD and varying percentages of modified flavins such as 6-OH-FAD and 8-OH-FAD. The additionally bound FAD shows an anomalous absorption spectrum with strong absorption around 400 nm. This spectral change is not due to a chemical modification of the flavin ring because the flavin released by KBr or guanidine hydrochloride is normal FAD. It is also not due to unknown small molecules because the same spectrum appears when ETF is reconstituted from its guanidine-denatured subunits and FAD. A similar anomalous spectrum was observed for AMP-free pig ETF under acidic conditions, suggesting a common flavin environment between pig and M. elsdenii ETFs.

Structure of the transition state analog of medium-chain acyl-CoA dehydrogenase. Crystallographic and molecular orbital studies on the charge-transfer complex of medium-chain acyl-CoA dehydrogenase with 3-thiaoctanoyl-CoA.

The flavoenzyme medium-chain acyl-CoA dehydrogenase (MCAD) eliminates the alpha-proton of the substrate analog, 3-thiaoctanoyl-CoA (3S-C8-CoA), to form a charge-transfer complex with deprotonated 3S-C8-CoA. This complex can simulate the metastable reaction intermediate immediately after the alpha-proton elimination of a substrate and before the beta-hydrogen transfer as a hydride, and is therefore regarded as a transition-state analog. The crystalline complex was obtained by co-crystallizing MCAD in the oxidized form with 3S-C8-CoA. The three-dimensional structure of the complex was solved by X-ray crystallography. The deprotonated 3S-C8-CoA was clearly located within the active-site cleft of the enzyme. The arrangement between the flavin ring and deprotonated 3S-C8-CoA is consistent with a charge transfer interaction with the negatively charged acyl-chain of 3S-C8-CoA as an electron donor stacking on the pyrimidine moiety of the flavin ring as an electron acceptor. The structure of the model complex between lumiflavin and the deprotonated ethylthioester of 3-thiabutanoic acid was optimized by molecular orbital calculations. The obtained theoretical structure was essentially the same as that of the corresponding region of the X-ray structure. A considerable amount of negative charge is transferred to the flavin ring system to stabilize the complex by 9.2 kcal/mol. The large stabilization energy by charge transfer probably plays an important role in determining the alignment of the flavin ring with 3S-C8-CoA. The structure of the highest occupied molecular orbital of the complex revealed the electron flow pathway from a substrate to the flavin ring.

Molecular mechanism of the drop in the pKa of a substrate analog bound to medium-chain acyl-CoA dehydrogenase: implications for substrate activation.

The pKa value of a substrate analogue 3-thiaoctanoyl-CoA at alphaC-H is known to drop from ca. 16 in the free state to 5-6 upon binding to medium-chain acyl-CoA dehydrogenase (MCAD). The molecular mechanism underlying this phenomenon was investigated by taking advantage of artificial FADs, i.e., 8-CN-, 7,8-Cl2-, 8-Cl-, 8-OCH3-, 8-NH2-, ribityl-2'-deoxy-8-CN-, and ribityl-2'-deoxy-8-Cl-FADs, reconstituted into MCAD. The stronger the electron-withdrawing ability of the substituent, the smaller the pKa value became [e.g., 7.4 (8-NH2-FAD) and 4.0 (8-CN-FAD)], suggesting that the flavin ring itself affects the pKa value of the ligand via a charge-transfer interaction with the ligand. The destruction of the hydrogen bond between the thioester C(1)=O and the ribityl-2'-OH of FAD raised the pKa by ca. 2.5 units. These results indicate that the interaction between the ligand and the flavin ring also serves to lower the pKa of the ligand, in addition to the hydrogen bonds at C(1)=O of the ligand.

Three-dimensional structure of the flavoenzyme acyl-CoA oxidase-II from rat liver, the peroxisomal counterpart of mitochondrial acyl-CoA dehydrogenase.

Acyl-CoA oxidase (ACO) catalyzes the first and rate-determining step of the peroxisomal beta-oxidation of fatty acids. The crystal structure of ACO-II, which is one of two forms of rat liver ACO (ACO-I and ACO-II), has been solved and refined to an R-factor of 20.6% at 2.2-A resolution. The enzyme is a homodimer, and the polypeptide chain of the subunit is folded into the N-terminal alpha-domain, beta-domain, and C-terminal alpha-domain. The X-ray analysis showed that the overall folding of ACO-II less C-terminal 221 residues is similar to that of medium-chain acyl-CoA dehydrogenase (MCAD). However, the N-terminal alpha- and beta-domains rotate by 13 with respect to the C-terminal alpha-domain compared with those in MCAD to give a long and large crevice that accommodates the cofactor FAD and the substrate acyl-CoA. FAD is bound to the crevice between the beta- and C-terminal domains with its adenosine diphosphate portion interacting extensively with the other subunit of the molecule. The flavin ring of FAD resides at the active site with its si-face attached to the beta-domain, and is surrounded by active-site residues in a mode similar to that found in MCAD. However, the residues have weak interactions with the flavin ring due to the loss of some of the important hydrogen bonds with the flavin ring found in MCAD. The catalytic residue Glu421 in the C-terminal alpha-domain seems to be too far away from the flavin ring to abstract the alpha-proton of the substrate acyl-CoA, suggesting that the C-terminal domain moves to close the active site upon substrate binding. The pyrimidine moiety of flavin is exposed to the solvent and can readily be attacked by molecular oxygen, while that in MCAD is protected from the solvent. The crevice for binding the fatty acyl chain is 28 A long and 6 A wide, large enough to accommodate the C23 acyl chain.

Effects of hydrogen bonds in association with flavin and substrate in flavoenzyme d-amino acid oxidase. The catalytic and structural roles of Gly313 and Thr317.

According to the three-dimensional structure of a porcine kidney D-amino acid oxidase-substrate (D-leucine) complex model, the G313 backbone carbonyl recognizes the substrate amino group by hydrogen bonding and the side-chain hydroxyl of T317 forms a hydrogen bond with C(2)=O of the flavin moiety of FAD [Miura et al. (1997) J. Biochem. 122, 825-833]. We have designed and expressed the G313A and T317A mutants and compared their enzymatic and spectroscopic properties with those of the wild type. The G313A mutant shows decreased activities to various D-amino acids, but the pattern of substrate specificity is different from that of the wild type. The results imply that the hydrogen bond between the G313 backbone carbonyl and the substrate amino group plays important roles in substrate recognition and in defining the substrate specificity of D-amino acid oxidase. The T317A mutant shows a decreased affinity for FAD. The steady-state kinetic measurements indicate diminished activities of T317A to substrate D-amino acids. The transient kinetic parameters measured by stopped-flow spectroscopy revealed that T317 plays key roles in stabilizing the purple intermediate, a requisite intermediate in the oxidative half-reaction, and in enhancing the release of the product from the active site, thereby optimizing the overall catalytic process of D-amino acid oxidase.