FAD binding in glycine oxidase from Bacillus subtilis

The apoprotein of the FAD-containing flavoenzyme glycine oxidase from Bacillus subtilis was obtained at pH 8.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M Tris–HCl and 20% glycerol. The apoprotein of glycine oxidase shows high protein fluorescence, high exposure of hydrophobic surfaces, and low temperature stability as compared to the holoenzyme. The isolated apoprotein species is present in solution as a monomer which rapidly recovers its tertiary structure and converts into the tetrameric holoenzyme following incubation with free FAD. The reconstitution process follows a particular two-stage process; the spectral properties of the reconstituted holoenzyme were virtually indistinguishable from those observed with native glycine oxidase, while the activity was only partially (50%) recovered. The urea-induced unfolding process of glycine oxidase can be considered as a two-step (three-state) process: the presence of intermediate(s) in the unfolding process of the holoenzyme at ≈2 M urea is evident in the changes of the flavin fluorescence intensity and can be also inferred from the different urea sensitivities of the spectral probes used. On the other hand, only a single transition at ≈4.5 M urea concentration is observed for the apoprotein form. The chemical denaturation of glycine oxidase holoenzyme is partially reversible (e.g., no activity is recovered when starting the refolding from 4 M urea-denatured holoprotein). Finally, the introduction by site-directed mutagenesis of residues corresponding to those involved in the covalent link with FAD in the related flavoenzyme monomeric sarcosine oxidase failed to convert glycine oxidase into a covalent flavoprotein. These investigations show that the consequences of FAD binding for the stability and folding process distinguish glycine oxidase from enzymes active on similar compounds.     

Effect of halide anions on the binding of FAD to D-amino acid oxidase and the tryptophanyl fluorescence of the apoenzyme.

The quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25 degrees C. All of the tryptophanyl fluorescence of the apoenzyme in 6 M guanidine hydrochloride was quenched. The tryptophanyl fluorescence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism. By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution containing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.     

The recognition of glycolate oxidase apoprotein with flavin analogs in higher plants

The net photosynthetic efficiency in C3 plants (such asrice, wheat and other major crops) can be decreased by30% due to the metabolism of photorespiration [1], inwhich glycolate oxidase (GO) serves as a key enzyme. Itis known that GO, with flavin mononucleotide (FMN) asa cofactor, belongs to flavin oxidase [2]. But it differs fromother flavoproteins in that FMN is loosely bound to itsapoprotein and there exists a dissociation balance betweenthem, which indicates that FMN probably regulate…     

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.     

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.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

Deflavination of flavo-oxidases by nucleophilic reagents

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.     

Effect of hydrophobic probes on the higher structure of D-amino acid oxidase.

1. The holoenzyme of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] was found to combine with 1-anilinonaphthalene-8-sulfonate without liberation of its coenzyme, FAD. No energy transfer interaction was found to occur between the bound dye and FAD of the holoenzyme. On the other hand, when the apoenzyme was bound to the dye and then to FAD, energy transfer interaction between the bound dye and bound FAD was observed. In both cases, the dye competes with the substrate, D-alanine. It is concluded that the dye bound to the holoenzyme is oriented in such a special manner that the mutual orientation factor between the dye and FAD becomes very small in magnitude. 2. When the apoenzyme combined with the dye, the monomer-dimer equilibrium of the apoenzyme shifted towards the dimer. On the other hand, 4-monobenzoylamido-4'-aminostilbene-2,2'-disulfonate combined with the apoenzyme to induce monomerization.     

Structure and function of D-amino acid oxidase. IX. Changes in the fluorescence polarization of FAD upon complex formation.

1. The fluorescence polarization, P, of FAD increased on complex formation with the apoenzyme of D-amino acid oxidase [D-amino acid: O2 ocidoreductase (deaminating), EC 1.4.3.3]. The time course of the increase was monophasic. The values of P were extimated to be 0.04, 0.4, and 0.4 for FAD, the enzyme and the enzyme-benzoate complex, respectively. 2. The value of P of the enzyme is dependent on its concentration, indicating that the degrees of dissociation of FAD in the monomer and dimer are different. The dissociation constant was calculated to be 7 times 10-minus 7 M for the monomeric form of the enzyme. This value is far larger than the value for the dimeric form of the enzyme, 1 times 10-minus 8 M, calculated from equilibrium dialysis data. 3. Changes in fluorescence polarization of the enzyme due to changes in solution pH or temperature can be explained in terms of the monomer-dimer equilibrium.     

Relevance of weak flavin binding in human D‐amino acid oxidase

In the brain, the human flavoprotein D ‐amino acid oxidase (hDAAO) is involved in the degradation of the gliotransmitter D ‐serine, an important modulator of NMDA‐receptor‐mediated neurotransmission; an increase in hDAAO activity (that yields a decrease in D ‐serine concentration) was recently proposed to be among the molecular mechanisms leading to the onset of schizophrenia susceptibility. This human flavoenzyme is a stable homodimer (even in the apoprotein form) that distinguishes from known D ‐amino acid oxidases because it shows the weakest interaction with the flavin cofactor in the free form. Instead, cofactor binding is significantly tighter in the presence of an active site ligand. In order to understand how hDAAO activity is modulated, we investigated the FAD binding process to the apoprotein moiety and compared the folding and stability properties of the holoenzyme and the apoprotein forms. The apoprotein of hDAAO can be distinguished from the holoenzyme form by the more “open” tertiary structure, higher protein fluorescence, larger exposure of hydrophobic surfaces, and higher sensitivity to proteolysis. Interestingly, the FAD binding only slightly increases the stability of hDAAO to denaturation by urea or temperature. Taken together, these results indicate that the weak cofactor binding is not related to protein (de)stabilization or oligomerization (as instead observed for the homologous enzyme from yeast) but rather should represent a means of modulating the activity of hDAAO. We propose that the absence in vivo of an active site ligand/substrate weakens the cofactor binding, yielding the inactive apoprotein form and thus avoiding excessive D ‐serine degradation.     

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.     

Characterization of a D-amino acid oxidase with high activity against cephalosporin C from the yeast Trigonopsis variabilis

We describe an improved method to purify D-amino acid oxidase with activity towards cephalosporin C. The protein has a carbohydrate content of 1.3% and two molecules of non-covalently bound flavin cofactor per protein molecule. HPLC profiles and enzymatic analysis have indicated that the cofactor is FAD, even though fluorescence spectroscopy shows a slightly altered spectral profile in the 400-500 nm range compared to authentic FAD. N-terminal sequencing of the protein revealed a high level of similarity (56% identity in 25 amino acids) between the fungal and mammalian oxidase, and probably represents a "Rossman fold" with a beta-alpha-beta structure for the binding of the adenosyl moiety of the cofactor.     

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.     

The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged D-amino acid oxidase from Trigonopsis variabilis

d-amino acid oxidase from Trigonopsis variabilis (TvDAAO) is a flavoenzyme with high biotechnological and industrial interest. The overexpression and purification of the apoprotein form of a recombinant His-tagged TvDAAO allowed us to go deep into the structural differences between apoenzyme and holoenzyme, and on the cofactor binding and its contribution to enzyme stability. A significant decrease in intrinsic fluorescence emission took place upon FAD binding, associated to cofactor induced conformational transitions or subunit dimerization that could affect the local environment of protein tryptophan residues. Furthermore, acrylamide-quenching experiments indicated that one of the five tryptophan residues of TvDAAO became less accessible upon FAD binding. A K(d)=1.5+/-0.1x10(-7) M for the dissociation of FAD from TvDAAO was calculated from binding experiments based on both quenching of FAD fluorescence and activity titration curves. Secondary structure prediction indicated that TvDAAO is a mixed alpha/beta protein with 8 alpha-helices and 14 beta-sheets connected by loops. Prediction results were in good agreement with the estimates obtained by circular dichroism which indicated that both the apoenzyme and the holoenzyme had the same structural component ratios: 34% alpha-helix content, 20% beta-structure content (14% antiparallel and 6% parallel beta-sheet), 15% beta-turns and 31% of random structure. Circular dichroism thermal-transition curves suggested single-step denaturation processes with apparent midpoint transition temperatures (T(m)) of 37.9 degrees C and 41.4 degrees C for the apoenzyme and the holoenzyme, respectively. A three-dimensional model of TvDAAO built by homology modelling and consistent with the spectroscopic studies is shown. Comparing our results with those reported for pig kidney (pkDAAO) and Rhodotorula gracilis (RgDAAO) d-amino acid oxidases, a "head-to-head" interaction between subunits in the TvDAAO dimer might be expected.     

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.     

Release of Flavin Adenine Dinucleotide in the Course of a Local Conformational Transition Induced by Trimethylamine Dehydrogenase in Electron-Transferring Flavoprotein

A pair of proteins involved in electron transfer, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro. It was demonstrated by fluorescence spectroscopy that flavin adenine dinucleotide (FAD) can slowly and spontaneously be released from ETF. This release is followed by increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is dramatically activated by TMAD preparations that induce a local conformational transition in ETF. It was shown on the basis of the values of tryptophan polarization and lifetime with the use of the Levshin–Perrin equation that the sizes of protein particles were not changed after mixing of TMAD and ETF; i.e., these proteins by themselves did not form a stable complex with each other. The release of flavin from ETF did not occur in the presence of trimethylamine and formaldehyde in the protein mixture. In this case, a stable complex between the proteins is probably formed with the participation of formaldehyde. FAD is hydrolyzed to flavin mononucleotide (FMN) and AMP after a short-term incubation of ETF with ferricyanide. This fact explains the previous detection of AMP in ETF preparations by other researches. A fluorescence method for distinguishing FAD from FMN in solution with the use ethylene glycol is proposed.     

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.     

A study of flavin-protein and flavoprotein-ligand interactions. Binding aspects and spectral properties of D-amino acid oxidase and riboflavin binding protein.

To study flavin-protein and flavoprotein-ligand interaction, the absorption, CD and MCD spectra of riboflavin, FAD, roseoflavin, the complexes of riboflavin and roseoflavin with riboflavin binding protein(RBP),D-amino acid oxidase(D-AO) and its complexes with ligands were observed in the spectral region of 310-600 nm and the binding properties of D-AO with di-substituted benzoate derivatives and of RBP with roseoflavin were also measured. The dimer of D-amino acid oxidase has a higher affinity for di-substituted benzoate derivatives than the monomer. The change in the absorption of FAD in D-AO caused by the binding of the first ligand to the dimer, which can bind two ligands, was similar to that caused by the binding of the second ligand. Roseoflavin could bind to RBP in a 1 : 1 ratio and the dissociation constant was 3.8 x 10(-8)M. The protein fluorescence of RBP was quenched by about 86% due to complex formation with roseoflavin. The MCD spectra showed similar patterns for all molecular complexes of riboflavin and FAD, with two negative extrema of ellipticity which probably correspond to the Faraday B-term, but the Faraday A-term could not be observed, suggesting that there was no degeneracy in the excited state of flavins. It is also suggested, based on a comparison of the absorption, CD and MCD spectra, that the vibronic structure of flavin was modified differently by each flavin-protein or flavoprotein-ligand interaction. Comparison of the absorption, CD and MCD spectra(310-600 nm) for roseoflavin and the roseoflavin-RBP complex revealed that there were five spectral components around 320, 340, 400, 500, and 550 nm in roseoflavin.     

Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme. The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks. In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined. FRG/FRase I followed a ping-pong kinetic mechanism. It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate. FAD and riboflavin were also active substrates. FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively. All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I. Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP.