A study of the interaction between D-amino acid oxidase and quasi-substrates.

To study the interaction between D-amino acid oxidase [EC 1.4.3.3] and quasi-substrates such as benzoate and o-, m-, and p-aminobenzoate, visible circular dichroism spectra (CD spectra) were measured and the binding rate and affinity of o-aminobenzoate to the enzyme were observed by following the absorption changes at various wavelengths. We found a new CD band around 560 nm, corresponding to the charge-transfer complexes which result from the formation of aminobenzoate complexes with the enzyme. The ellipticity of this band was positive for the p-aminobenzoate complex, but negative for the o- and m-aminobenzoate complexes. Crossover points in CD spectra were observed at 470 nm for the m-aminobenzoate complex and at 475 nm for the o-aminobenzoate complex. They probably resulted from overlapping of the positive CD band of FAD bound with the enzyme and the negative CD band of the charge-transfer complex. We propose that the amino group in aminobenzoate, not the pi-electrons of the benzene ring, is the electron donor in the charge-transfer complex and that the position of the amino group is very important for the charge-transfer interaction. The binding rate and affinity of o-aminobenzoate to the enzyme were determined using the absorption changes at 370 nm (380 nm), caused by the modification of electronic states of FAD bound with the enzyme, and at 550 nm (565 nm), caused by the formation of the charge-transfer complex of o-aminobenzoate with the enzyme. No differences between these parameters with wavelength were observed. This independence of wavelength simplifies discussion of the experimental data obtained from absorption changes.     

The bifunctional flavokinase/flavin adenine dinucleotide synthetase from Streptomyces davawensis produces inactive flavin cofactors and is not involved in resistance to the antibiotic roseoflavin

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5'-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, kcat/Km = 1.7 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 7.5 10(-3) microM(-1) s(-1)). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1)). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.     

Infrared magnetic circular dichroism of myoglobin derivatives.

By use of a newly constructed CD instrument, infrared magnetic circular dichroism (MCD) spectra were observed for various myoglobin derivatives. The ferric high spin myoglobin derivatives such as fluoride, water and hydroxide complexes, commonly exhibited the MCD spectra consisting of positive A terms. Therefore, the results reinforced the assignment that the infrared band is the charge transfer transition to the degenerate excited state (eg (dpi)). Since the fraction of A term estimated was approximately 80% for myoglobin fluoride and approximately 35% for myoglobin water, the effective symmetry for myoglobin fluoride is determined to be as close as D4h, while that for myoglobin water seems to have lower symmetry components. The ferric low spin derivatives such as myoglobin cyanide, myoglobin imidazole and myoglobin azide showed positive MCD spectra which are very similar to the electronic absorption spectra. These MCD spectra were assigned to the charge transfer transitions from porphyrin pi to iron d orbitals on the ground that they were observed only for the ferric low spin groups and insensitive to the axial ligands. The lack of temperature dependence in the MCD magnitude indicated that the MCD spectra are attributable to the Faraday B terms. Deoxymyoglobin, the ferrous high spin derivative, had fairly strong positive MCD around 760 nm with an anisotropy factor (delta epsilon/epsilon) of 1.4-10(-4). It shows some small MCD bands from 800 to 1800 nm. Among the ferrous low spin derivatives, carbonmonoxymyoglobin did not give any observable MCD in the infrared region while oxymyoglobin seemed to have significant MCD in the range from 700 to 1000 nm.     

Proton release from flavoprotein D-amino acid oxidase on complexation with the zwitterionic ligand, trigonelline

Trigonelline, i.e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.     

Covalent flavinylation of vanillyl-alcohol oxidase is an autocatalytic process

Vanillyl-alcohol oxidase (VAO; EC 1.1.3.38) contains a covalently 8alpha-histidyl bound FAD, which represents the most frequently encountered covalent flavin-protein linkage. To elucidate the mechanism by which VAO covalently incorporates the FAD cofactor, apo VAO was produced by using a riboflavin auxotrophic Escherichia coli strain. Incubation of apo VAO with FAD resulted in full restoration of enzyme activity. The rate of activity restoration was dependent on FAD concentration, displaying a hyperbolic relationship (K(FAD )= 2.3 microM, k(activation) = 0.13 min(-1)). The time-dependent increase in enzyme activity was accompanied by full covalent incorporation of FAD, as determined by SDS/PAGE and ESI-MS analysis. The results obtained show that formation of the covalent flavin-protein bond is an autocatalytic process, which proceeds via a reduced flavin intermediate. Furthermore, ESI-MS experiments revealed that, although apo VAO mainly exists as monomers and dimers, FAD binding promotes the formation of VAO dimers and octamers. Tandem ESI-MS experiments revealed that octamerization is not dependent on full covalent flavinylation.     

Temperature-induced changes in the coenzyme environment of D-amino acid oxidase revealed by the multiple decays of FAD fluorescence.

A temperature-dependent change in the microenvironment of the coenzyme, FAD, of D-amino acid oxidase was investigated by means of steady-state and picosecond time-resolved fluorescence spectroscopy. Relative emission quantum yields from FAD bound to D-amino acid oxidase revealed the temperature transition when concentration of the enzyme was lowered. The observed fluorescence decay curves were well described with four-exponential decay functions. The amplitude of the shortest lifetime (tau 0), approximately 25 ps, was always negative, which indicates that the fluorescence of D-amino acid oxidase at approximately 520 nm appears after a metastable state of the excited isoalloxazine decays. The other components with positive amplitudes were assigned to dimer or associated forms of the enzyme, monomer, and free FAD dissociated from the enzyme. Ethalpy and entropy changes of intermediate states in the quenching processes were evaluated according to the absolute rate theory. The temperature transition was much more pronounced in the monomer than in the dimer or associated forms of the enzyme.     

Electronic state of heme in cytochrome oxidase. I. Magnetic circular dichroism of the isolated enzyme and its derivatives.

Magnetic circular dichroism (MCD) spectra have been recorded for beef heart cytochrome oxidase and a number of its inhibitor complexes. The resting enzyme exhibits a derivate shape Faraday C term in the Soret region, characteristic of low spin ferric heme, which accounts for 50% of the total oxidase heme a. The remaining heme a (50%) is assigned to the high spin state. MCD temperature studies, comparison with the MCD spectra of heme a-imidazole model compounds, and ligand binding (cyanide, formate) studies are consistent with these spin state assignments in the oxidized enzyme. Furthermore, the ligand binding properties and correlations between optical and MCD parameters indicate that in the resting enzyme the low spin heme a is due solely to cytochrome a3+ and the high spin heme a to cytochrome a33+. The Soret MCD of the reduced protein is interpreted as th sum of two MCD curves: an intense, asymmetric MCD band very similar to that exhibited by deoxymyoglobin which we assign to paramagnetic high spin cytochrome a3(2+) and a weaker, more symmetric MCD contribution, which is attributed to diamagnetic low spin cytochrome a2+. Temperature studies of the Soret MCD intensity support this proposed spin state heterogeneity. Ligand binding (CO, CN-) to the reduced protein eliminates the intense MCD associated with high spin cytochrome a3(2+); however, the band associated with cytochrome a2+ is observed under these conditions as well as in a number of inhibitor complexes (cyanide, formate, sulfide, azide) of the partially reduced protein. The MCD spectra of oxidized, reduced, and inhibitor-complexed cytochrome oxidase show no evidence for heme-heme interaction via spectral parameters. This conclusion is used in conjunction with the fact that ferric, high spin heme exhibits weak MCD intensity to calculate the MCD spectra for the individual cytochromes of the oxidase as well as the spectra for some inhibitor complexes of cytochrome a3. The results are most simply interpreted using the model we have recently proposed to account for the electronic and magnetic properties of cytochrome (Palmer, G., Babcock, F.T., and Vcikery, L.E. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 2206-2210).     

Exchange of free and bound coenzyme of flavin enzymes studied with [14C]FAD.

The exchange of bound FAD for free FAD was studied with D-amino acid oxidase (D-amino acid:oxygen oxidoreductase (deaminating), EC 1.4.3.3) and beta-D-glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4). For a simple measurement of the reaction rate, equimolar amounts of the enzyme and [14C]FAD were mixed. The exchange occurred very rapidly in the holoenzyme of D-amino acid oxidase at 25 degrees C, pH 8.3 (half life of the exchange: 0.8 min), but slowly in the presence of the substrate or a competitive inhibitor, benzoate. It also occurred slowly in the purple complex of D-amino acid oxidase. In the case of beta-D-glucose oxidase, however, the exchange occurred very slowly at 25 degrees C, pH 5.6, regardless of the presence of the substrate or p-chloromercuribenzoate. On the basis of these findings, the turnover of the coenzymes of flavin enzymes in mammals is discussed.     

Affinity labeling of D-amino acid oxidase with an acetylenic substrate.

The acetylenic substrate, D-2-amino-4-pentynoic acid (D-propargylglycine), was oxidatively deaminated by hog kidney D-amino acid oxidase[EC 1.4.3.3], with accompanying inactivation of the enzyme. The flavin which was extracted by hot methanol from the inactivated enzyme was identical with authentic FAD by thin-layer chromatography and circular dichroism. The excitation spectrum of emission at 520 nm of the released flavin was very similar to the absorption spectrum of oxidized FAD. The released flavin was reduced by potassium borohydride. The apoenzyme prepared after propargylglycine treatment did not show restored D-amino acid oxidase activity on adding exogenous FAD. The absorption spectrum of this inactivated apoenzyme showed absorption peaks at 279 and 317 nm, and a shoulder at about 290 nm. These results strongly indicate that the inactivation reaction is a dynamic affinity labeling with D-propargylglycine which produces irreversible inactivation of the enzyme by a covalent modification of an amino acid residue at the active site.     

Magnetic circular dichroism study of cytochrome ba3 from Thermus thermophilus: spectral contributions from cytochromes b and a3 and nanosecond spectroscopy of CO photodissociation intermediates.

Near-UV-vis magnetic and natural circular dichroism (MCD and CD) spectra of oxidized, reduced, and carbonmonoxy-complexed cytochrome ba3, a terminal oxidase from the bacterium Thermus thermophilus, and nanosecond time-resolved MCD (TRMCD) and CD (TRCD) spectra of the unligated species formed after photodissociation of the CO complex are presented. The spectral contributions of individual cytochromes b and a3 to the Soret region MCD are identified. TRMCD spectroscopy is used to follow the spin state change (S = 0 to S = 2) in cytochrome a3(2+) following photodissociation of the CO complex. There is prompt appearance of the high-spin state after photolysis, as found previously in mammalian cytochrome oxidase [Goldbeck, R. A., Dawes, T. D., Einarsdóttir, O., Woodruff, W. H., & Kliger, D. S. (1991) Biophys. J. 60, 125-134]. Peak shifts of 1-10 nm appear in the TRMCD, TRCD, and time-resolved UV-vis absorption spectra of the photolyzed enzyme throughout its observable lifetime, indicating that the photolyzed enzyme does not relax to its equilibrium deliganded form before recombination with CO occurs hundreds of milliseconds later. Direct heme-heme interaction is not found in cytochrome ba3, but red-shifts in the MCD and absorption spectra of both cytochromes b and (photolyzed) a3 are correlated with a CO-liganded form of the protein. The long time (tau approximately greater than 1 s) needed for relaxation of the cytochrome b and a3 peaks to their static positions suggests that CO binding to a3 induces a global conformational change in the protein that weakly perturbs the MCD and absorption spectra of b and photolyzed a3. Fea3 binds CO more weakly in cytochrome ba3 than in cytochrome aa3. The MCD spectrum of reduced enzyme solution placed under 1 atm of CO contains a peak at 446 nm that shows approximately 30% of total cytochrome a3 remains pentacoordinate, high-spin.     

Interaction of anions with the active site of carboxypeptidase A

Studies of azide inhibition of peptide hydrolysis catalyzed by cobalt(II) carboxypeptidase A identify two anion binding sites. Azide binding to the first site (KI = 35 mM) inhibits peptide hydrolysis in a partial competitive mode while binding at the second site (KI = 1.5 M) results in competitive inhibition. The cobalt electronic absorption spectrum is insensitive to azide binding at the first site but shows marked changes upon azide binding to the second site. Thus, azide elicits a spectral change with new lambda max (epsilon M) values of 590 (330) and 540 nm (190) and a KD of 1.4 M, equal to the second kinetic KI value for the cobalt enzyme, indicating that anion binding at the weaker site involves an interaction with the active-site metal. Remarkably, in the presence of the C-terminal products of peptide or ester hydrolysis or carboxylate inhibitor analogues, anion (e.g., azide, cyanate, and thiocyanate) binding is strongly synergistic; thus, KD for azide decreases to 4 mM in the presence of L-phenylalanine. These ternary complexes have characteristic absorption, CD, MCD, and EPR spectra. The absorption spectra of azide/carboxylate inhibitor ternary complexes with Co(II)CPD display a near-UV band between 305 and 310 nm with epsilon M values around 900-1250 M-1 cm-1. The lambda max values are close to the those of the charge-transfer band of an aquo Co(II)-azide complex (310 nm), consistent with the presence of a metal azide bond in the enzyme complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Picosecond laser fluorometry of FAD of D-amino acid oxidase-benzoate complex

Formation of a complex of D-amino acid oxidase (D-amino acid:O2 oxidoreductase (deaminating), EC 1.4.3.3) and benzoate, an enzyme-substrate complex model, was studied by measuring the fluorescence life-time of the coenzyme FAD of the complex by using a mode-locked Nd:YAG laser and a streak camera. The value of lifetime was 60 +/- 10 ps in the monomer of the complex and it was extremely short (much less than 5 ps) in the dimer of the complex. Since the values of fluorescence lifetime of the coenzyme are 130 ps in the monomeric form of free enzyme and 40 ps in the dimeric form of free enzyme, the decrease in the lifetime upon complex formation with benzoate is slight in the monomer (reduced to one-half) whereas marked in the dimer (reduced to less than 1/10). By analyzing the fluorescence decay curve, a dissociation constant of the monomer-dimer equilibrium of the complex was evaluated to be 0.4 +/- 0.3 microM, which is much smaller than that in free enzyme. Fluorescence analysis under steady state excitation revealed that the apparent dissociation constant (K) of FAD from the enzyme was decreased by 1:1000 upon the complex formation. Relative quantum yield of the fluorescence of FAD in the complex to that of free FAD exhibited appreciable dependence on the complex concentration: greater in the monomer and less in the dimer. These results suggest that a molecular interaction between FAD and amino acid residue(s) is strengthened by the complex formation, which contributes to a remarkable conformational change in the protein moiety of the complex.     

Magnetic circular dichroism studies of bovine liver catalase.

Absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of beef liver catalase at pH 5.0 and 6.9, and its complexes with NaF, KCNO, NaCNS, NaN3 and NaCN, have been measured between 250 nm and 700 nm at room temperature. The pH 6.9 native catalase MCD shows the presence of several additional transitions not resolved in the absorption spectrum. While these bands can be seen in the spectra of all the derivatives, with the exception of the cyanide, their relative intensities changes considerably between complexes. Of special interest in the MCD of ferric hemes is the signal intensity at about 400 nm and 620 nm. The data indicate that the MCD intensity at 620 nm increases as the high spin iron porphyrin fraction increases, reaching a maximum with the fluoride complex. The 430 nm band intensity increases as the proportion of low spin iron increases, reaching a maximum with the cyanide complex. The MCD spectra also indicate clearly the existence of spin mixtures in the complexes with CNO-, CNS-, and N3-, where both the 430 nm and 620 nm bands have appreciable intensity. It is significant that despite almost identical absorption spectra the CNS- complex has higher fraction of low spin iron than either the CNO- or the N3- species. The differences between the pH 5 and 6.9 MCD spectra of the native catalase suggest that the environment of the heme centre is sensitive to protonation.     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Thermodynamic control of D-amino acid oxidase by benzoate binding

The redox properties of D-amino acid oxidase (D-amino-acid: O2 oxidoreductase (deaminating) EC1.4.3.3) have been measured at 18 degrees C in 20 mM sodium pyrophosphate, pH 8.5, and in 50 mM sodium phosphate, pH 7.0. Over the entire pH range, 2 eq are required per mol of FAD in D-amino acid oxidase for reduction to the anion dihydroquinone. The red anion semiquinone is thermodynamically stable as indicated by the separation of the electron potentials and the quantitative formation of the semiquinone species. The first electron potential is pH-independent at -0.098 +/- 0.004 V versus SHE while the second electron potential is pH-dependent exhibiting a 0.060 mV/pH unit slope. The redox behavior of D-amino acid oxidase is consistent with that observed for other oxidase enzymes. On the other hand, the behavior of the benzoate-bound enzyme under the same conditions is in marked contrast to the thermodynamics of free D-amino acid oxidase. Spectroelectrochemical experiments performed on inhibitor-bound (benzoate) D-amino acid oxidase show that benzoate binding regulates the redox properties of the enzyme, causing the energy levels of the benzoate-bound enzyme to be consistent with the two-electron transfer catalytic function of the enzyme. Our data are consistent with benzoate binding at the enzyme active site destroying the inductive effect of the positively charged arginine residue. Others have postulated that this positively charged group near the N(1)C(2) = O position of the flavin controls the enzyme properties. The data presented here are the clearest examples yet of enzyme regulation by substrate which may be a general characteristic of all flavoprotein oxidases.     

8-thiocyanatoflavins as active-site probes for flavoproteins.

8-Thiocyanatoflavins at the riboflavin, FMN, and FAD level were prepared via the diazonium salt of the corresponding 8-aminoflavin and some of the physical and chemical properties studied. 8-Thiocyanatoriboflavin has a UV-visible spectrum similar to that of the native flavin with absorbance maxima at 446 nm (epsilon = 14,900 M-1 cm-1) and 360 nm. Reaction with thiols such as dithiothreitol and mercaptoethanol gives rise to an 8-mercapto- and an 8-SR-flavin, whereas reaction with sulfide yields only the 8-mercaptoflavin. The 8-SCN-flavin binds to riboflavin-binding protein as the riboflavin derivative, to apoflavodoxin, apo-Old Yellow Enzyme, and apo-lactate oxidase as the FMN derivative, and to apo-D-amino acid oxidase, apo-p-hydroxybenzoate hydroxylase, apo-glucose oxidase, apo-anthranilate hydroxylase, and apo-general acyl-CoA dehydrogenase as the FAD derivative. In two cases, namely, with anthranilate hydroxylase and D-amino acid oxidase, the 8-SCN-FAD was spontaneously and completely converted to the 8-mercapto-FAD derivative, suggesting the presence of a nucleophile (most likely the thiol of a cysteine residue) in the vicinity of the 8-position. It was also found that flavodoxin stabilizes the neutral radical and Old Yellow Enzyme the anionic radical of 8-SCN-FMN. Further studies with Old Yellow Enzyme, established that fully (two electron) reduced 8-SCN-FMN undergoes photoelimination of cyanide.     

A ligand field model for MCD spectra of biological cupric complexes.

A ligand field calculation of magnetic circular dichroism (MCD) spectra is described that provides new insights into the information contained in electronic spectra of copper sites in metalloenzymes and synthetic analogs. The ligand field model uses metal-centered p- and f-orbitals to model sigma, pi LMCT mixing mechanism for intensity, allowing the basic features of optical absorption, MCD, and electron paramagnetic resonance spectra to be simultaneously computed from a single set of parameters and the crystallographically determined ligand coordinates. We have used the model to predict changes in spectra resulting from the transformation of electronic wavefunctions under systematic variation in geometry in pentacoordinate ML5 complexes. The effectiveness of the calculation is demonstrated for two synthetic copper model compounds and a galactose oxidase enzyme complex representing limiting coordination geometries. This analysis permits immediate recognition of characteristic patterns of MCD intensity and correlation with geometry. A complementarity principle between MCD and CD spectra of transition metal complexes is discussed.     

Flavin binding to the high affinity riboflavin transporter RibU

The first biochemical and spectroscopic characterization of a purified membrane transporter for riboflavin (vitamin B(2)) is presented. The riboflavin transporter RibU from the bacterium Lactococcus lactis was overexpressed, solubilized, and purified. The purified transporter was bright yellow when the cells had been cultured in rich medium. We used a detergent-compatible matrix-assisted laser desorption ionization time-of-flight mass spectrometry method (Cadene, M., and Chait, B. T. (2000) Anal. Chem. 72, 5655-5658) to show that the source of the yellow color was riboflavin that had been co-purified with the transporter. The method appears generally applicable for substrate identification of purified membrane proteins. Substrate-free RibU was produced by expressing the protein in cells cultured in chemically defined medium. Riboflavin, FMN, and roseoflavin bound to RibU with high affinity and 1:1 stoichiometry (K(d) for riboflavin is 0.6 nM), but FAD did not bind to the transporter. The absorption spectrum of riboflavin changed dramatically when the substrate bound to RibU. Well resolved bands appeared at 441, 464, and 486 nm, indicating a hydrophobic binding pocket. The fluorescence of riboflavin was almost completely quenched upon binding to RibU, and also the tryptophan fluorescence of the transporter was quenched when flavins bound. The results indicate that riboflavin is stacked with one or more tryptophan residues in the binding pocket of RibU. Mutagenesis experiments showed that Trp-68 was involved directly in the riboflavin binding. The structural properties of the binding site and mechanistic consequences of the exceptionally high affinity of RibU for its substrate are discussed in relation to soluble riboflavin-binding proteins of known structure.     

A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase.

The apoenzyme of D-amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo-D-amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 x 10(-8) M was calculated. The kinetics of formation of the apoprotein-FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two-stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4-6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast D-amino acid oxidase appears to be of the type Apo + FAD in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.     

Resonance Raman study of D-amino acid oxidase-inhibitor complexes

The resonance Raman (RR) spectra of the complexes of D-amino acid oxidase (DAO) with benzoate derivatives were measured. The RR spectra of complexes of DAO with benzoate derivatives excited at 514.5 nm are similar to one another and also similar to that of oxidized flavin. In the cases of DAO-o-NH2-benzoate and DAO-o-OH-benzoate complexes, however, the line at 568 or 565 cm-1, derived from the benzoate derivative, was intensified. In the case of DAO-o-NH2-benzoate complex, which has an intense charge-transfer absorption band, the resonance enhancement of the Raman lines at 1583 and 568 cm-1 in the RR spectrum excited at 632.8 nm is striking. The former line is known to involve the vibrational displacements of the N(5) and C(4a) atoms of isoalloxazine and the latter is considered to be derived from a ring deformation mode of o-NH2-benzoate. This suggests that the o-NH2-benzoate molecule lies along the N(5)-C(4a) bond and parallel to the flavin face. A Raman line derived from o-OH-benzoate in the RR spectrum of DAO-o-OH-benzoate complex excited at 514.5 nm was detected. This result supports the view that the complex has a charge-transfer band, as has been pointed out by Massey and Ganther. Also, the spectrum of quasi-DAO-o-OH-benzoate complex is identical with that of the complex of DAO, suggesting that the active sites of these two enzymes have similar structures.