Covalent adducts of lactate oxidase. Photochemical formation and structure identification.

Lactate oxidase forms tight complexes with a variety of mono- and dicarboxylic acids. Most of these undergo facile photoreactions involving decarboxylation of the carboxylic acid and formation of covalent adducts at position N(5) of the flavin, characterized by absorption maxima from 325 to 365 nm and fluorescence emission in the range 440 to 490 nm. The properties of the adducts are strongly dependent on the nature of the substituent. Enzyme-bound N(5)-acyl adducts and N(5)-CH2-R derivatives are stable in the dark. Glycollyl- and alpha-lactyl adducts, however, decay to oxidized enzyme with half-lives in the order of minutes. Upon denaturation of the enzyme, the N(5)-alkyl adducts decay rapidly or are oxidized by oxygen. Reduced lactate oxidase is also photoalkylated in the presence of halogenated carboxylic acids. Bromoacetate yields an N(5)-carboxymethyl adduct; with beta-bromopropionate, a C(4a)-beta-propionyl derivate is formed. The N(5) adduct is identical with that from the photochemical reaction of oxidized enzyme and malonic acid. When the native coenzyme FMN is substituted by 2-S-FMN, qualitatively the same photoproducts are formed. The adducts obtained with the 2-S-FMN enzyme show the expected bathochromic shifts in absorption spectra. The results indicate that the photoreactivity of the enzyme is restricted to the positions C(4a) and N(5) of the flavin.     

Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.     

Resonance Raman spectra of anionic semiquinoid form of a flavoenzyme, D-amino acid oxidase

Resonance Raman (RR) spectra of the complex of anionic semiquinoid D-amino acid oxidase (DAO) with picolinate in H2O and D2O were observed in the 300-1,750 cm-1 region. RR spectra were also measured for the complex of the semiquinoid enzyme reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]-FAD. On the basis of the isotope effects, tentative assignments of the observed bands of the anionic semiquinoid flavin were made. The spectra differ from those of oxidized, neutral semiquinoid, and anionic reduced flavins previously reported. The 1,602 cm-1 band was not shifted for any FAD labeled in ring II and/or ring III and was assigned to a ring I mode. The 1,516 cm-1 band underwent an isotopic shift upon [4a-13C]- or [4,10a-13C2]-labeling. The band was assigned to the mode containing C(4a)-C(10a) stretching. The 1,331 and 1,292 cm-1 bands shifted upon [4a-13C]- or [5-15N]-labeling and were assigned to the modes containing C(4a)-N(5) stretching. The 1,217 and 1,188 cm-1 bands were assigned to the skeletal vibrations of ring III coupled with the N(3)-H bending mode. The RR spectrum of the complex of anionic semiquinoid DAO with alpha-iminopropionate or N-methyl-alpha-iminopropionate was essentially identical with that of the complex with picolinate.     

Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD.

Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillyl-alcohol oxidation. The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.     

Interaction of flavins with electron-rich metals.

A complex of the electron-rich ion Cu(I) with the flavoquinone analogue 10-methylisoalloxazine has been synthesized and characterized by x-ray methods. The complex is unstable to oxygen. It is black-green in color, in contrast with the bright yellow, orange, or orange-brown crystalline complexes of 10-methylisoalloxazine or riboflavin with Cu(II), Ag(I), and Pb(II). These results are indicative of strong perturbation of the flavin electronic structure by the Cu(I) ion and suggest that this complex is a reasonable model for incipient transfer of an electron from a reduced metal to flavoquinone. the crystal structure is orthorhombic, Pna2-1, with unit cell constants a = 31.24(1) (figures in parentheses are estimated standard deviations), b = 12.862(4), c = 6.239(2) A, Pobs = 1.76 g per cm-3 and Pcalc = 1.77 g per cm-3 for Z = 4 and asymmetric formula CuClO4-2(C11H8N4O2). HCOOH. The final R factor based on 1250 counter-measured data is 8.8%. The 2 independent 10-methylisoalloxazine molecules, A and B, bind strongly to the cuprous ion throug N(5) of each flavin. The copper is approximately linearly coordinated with an N-Cu-N angle of 153(1) degrees, and Cu-N(5) distances of 1.94(2) A and 1.92(2) A. The next nearest atoms to Cu are the O(4) oxygens of each flavin, forming weak bonds with distances Cu-O(4) = 2.27(2) A and 2.21(2) A for molecules A and B. The dihedral angle between the 2 10-methylisoalloxazine molecules is 65.4 degrees.     

Spectrometric evidence for the flavin-1-phenylcyclopropylamine inactivator adduct with monoamine oxidase N

1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with 1-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N(5)-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin-inactivator adduct. The incorporation of two (13)C labels into the inactivator, [2,3-(13)C(2)]-1-PCPA, followed by analysis using on-line liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin-inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N(5)-3-oxo-3-phenylpropyl-FAD.     

Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other β-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.     

Spectroscopic studies of pyridoxamine (pyridoxine) 5'-phosphate oxidase. Equilibrium dissociation constants and spectra for riboflavin 5'-phosphate and analogues.

Pyridoxamine (pyridoxine) 5'-phosphate oxidase (EC 1.4.3.5) has been shown to bind 1 mol of riboflavin 5'-phosphate (FMN) per mol of apoenzyme and is active with or inhibited by numerous FMN analogues [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436--3442]. The KD values and spectra for selected apoenzyme--flavin complexes have been determined and used to elucidate some of the properties of the FMN-binding site of this flavoprotein. Alterations of the pyrimidinoid portion of the flavin ring decrease binding considerably. The absorption spectra for the protein complexes with 3-deaza-FMN and 8-hydroxy-FMN indicate the presence of a dipolar or positively charged protein group near N1 and O2. The substitution of methyl for hydrogen at N3 apparently causes distortion of the interaction between the flavin ring and an active-site aromatic amino acid residue. Although binding is also decreased somewhat by substitutions at postions 8 and 8 alpha, considerable bulk [e.g., 8-(diethylamino)-FMN and 8 alpha-S-(N-acetyl-cysteinyl)-FMN] is accommodated. Hence, this portion of the flavin ring is probably oriented toward, possibly in contact with, solvent, as has been found for the flavodoxins. The importance of optimum interactions between the flavin and the apoprotein is further emphasized by large differences in the activity of flavin analogues that have similar midpoint potentials in solution.     

Mechanism of action of methanol oxidase, reconstitution of methanol oxidase with 5-deazaflavin, and inactivation of methanol oxidase by cyclopropanol

Methanol oxidase isolated from Hansenula polymorpha contains two distinct flavin cofactors in approximately equal amounts. One has been identified as authentic FAD and the other as a modified form of FAD differing only in the ribityl portion of the ribityldiphosphoadenosine side chain. The significance of this finding is as yet unknown. Previous studies have shown that cyclopropanol irreversibly inactivates methanol oxidase [Mincey, T., Tayrien, G., Mildvan, A. S., & Abeles, R. H. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7099-7101]. We have now established that inactivation is accompanied by covalent modification of the flavin cofactor. The stoichiometry of this reaction is 1 mol of cyclopropanol/mol of active flavin. The structure of the covalent adduct was determined by NMR, IR, and UV spectral studies to be an N5,C4a-cyclic 4a,5-dihydroflavin. Reduction of the covalent adduct with NaBH4 at pH 9.0 before removal from the enzyme converted it to the 1-(ribityldiphosphoadenosine)-substituted 4-(3-hydroxypropyl)-2,3-dioxoquinoxaline. Cyclopropyl ring cleavage accompanies inactivation, and covalent bond formation occurs between a methylene carbon of cyclopropanol and N5 of flavin. Methanol oxidase was also reconstituted with 5-deazaflavin adenine dinucleotide (dFAD). Reconstituted enzyme did not catalyze the oxidation of alcohols to the corresponding aldehydes, nor did reduced reconstituted enzyme catalyze the reverse reaction. Incubation of reconstituted enzyme with cyclopropanol resulted in an absorbance decrease at 399 nm, but no irreversible covalent modification of the deazaflavin cofactor. A reversible addition complex between cyclopropanol and dFAD is formed. The structure of that complex was not definitively established, but it is likely that it is formed through the addition of cyclopropoxide to C5 of dFAD. The failure of dFAD-reconstituted methanol oxidase to catalyze the oxidation of substrate, as well as the lack of reaction with cyclopropanol, supports a radical mechanism for alcohol oxidation and cyclopropanol inactivation. Methanol oxidase catalyzes the oxidation of cyclopropylcarbinol to the corresponding aldehyde. No ring-opened products were detected. The failure to form ring-opened products has been used as an argument against radical processes [MacInnes, I., Nonhebel, D. C., Orsculik, S. T., & Suckling, C. J. (1982) J. Chem. Soc., Chem. Commun., 121-122]. We present arguments against this interpretation.     

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.     

Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate] 2-hydroxy-3-butynoate.

2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.     

Inactivation and modification of lactate oxidase with fluorodinitrobenzene.

1. Dinitrophenylation of 2 +/- 0.2mol of residues/mol of enzyme-bound FMN resulted in the complete inactivation of the flavoenzyme L-lactate oxidase. 2. Hydrolysates of the inactivated enzyme contained 1mol each of Nim-Dnp-histidine (abbreviation: Dnp-,2,4-dinitrophenyl-; Nim indicates that either of the N atoms in the imidazole ring is substituted) and epsilon-Dnp-lysine/mol of FMN. 3. Competitive inhibitors decreased the extent of inactivation to a 10% loss of activity, and dinitrophenylation was decreased from 2 to approx. 0.5mol/mol of FMN. Only Nim-Dnp-histidine was detected in the hydrolysates. 4. Although the dinitrophenylated enzyme did not possess enzyme activitiy, L-lactate reduced approx. 50% of the enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme formed a complex with bisulphite. 6. The modified enzyme (2mol of Dnp/mol of FMN) was unable to bind substrate analogues and competitive inhibitors.     

Complex formation between anionic semiquinoid form of a flavoenzyme D-amino acid oxidase and ligands. Stabilizing mechanism of anionic semiquinoid flavoenzyme

Picolinate binds to the anionic semiquinoid form of D-amino acid oxidase (DAO), and the complex formed has a broad absorption band in the long-wavelength region extending beyond 800 nm, which is reminiscent of a charge transfer interaction. The binding has a stoichiometry of 1:1 with respect to the enzyme. The dissociation constant at 25 degrees C was 30 microM at pH 7.0. The pH dependence (pH 7.0-8.3) of the dissociation constant indicates that one proton is associated with the complex formation, and suggests that picolinate able to bind to the anionic semiquinoid enzyme is in the cationic form protonated at the nitrogen atom. By adding dithionite to the oxidized DAO solution containing pyruvate and various amines, a similar anionic semiquinoid DAO complex having a broad long-wavelength absorption band, appeared. Resonance Raman spectra with excitation at 623.8 nm of the anionic semiquinoid DAO complex formed in the presence of pyruvate and methylamine indicate that the complex consists of the anionic semiquinoid DAO and N-methyl-alpha-iminopropionate produced from pyruvate and methylamine, and that the imino group must be protonated. This supports the proposal that the presence of a positively charged group in the vicinity of flavin is required for the stabilization of the anionic semiquinoid flavin. The results also suggest that the broad absorption band is derived from the charge transfer interaction between the anionic semiquinoid flavin and the imino acid, in which the flavin C(4a)-N(5) locus and the locus containing (Formula: see text) of the amino acid are important for the interaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Resonance Raman study on the flavin in the purple intermediates of D-amino acid oxidase

Resonance Raman (RR) spectra were measured for the purple intermediates of D-amino acid oxidase reconstituted with isotopically labelled FAD's, i.e., [4a-13C]-, [4,10a-13C2]-, [2-13C]-, [5-15N]-, and [1,3-15N2]flavin adenine dinucleotides, and compared with those with the native enzyme. The RR lines around 1605 cm-1 with D-alanine or D-proline as a substrate and at 1548 cm-1 with D-alanine undergo isotopic shifts upon [4a-13C]- and [4,10a-13C2]-labelling. These lines are assigned to the vibrational modes associated with C(10a) = C(4a) - C(4) = O moiety of reduced flavin, providing the first assignment of RR lines of reduced flavin and conclusive evidence that reduced flavin is involved in this intermediate.     

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Active-site structural comparison of streptococcal NADH peroxidase and NADH oxidase. Reconstitution with artificial flavins.

The apoproteins of the streptococcal NADH peroxidase (H2O2----2H2O) and NADH oxidase (O2----2H2O) stabilize the neutral forms of 6-hydroxy- and 6-mercapto-FAD, respectively. The redox behavior of the 6-hydroxy-FAD peroxidase closely mimics that of the native enzyme with both dithionite and NADH. Both oxidase and peroxidase preferentially stabilize the N(1)-protonated p-quinonoid species of 8-mercapto-FAD, and the 8-position of the bound flavin is accessible to solvent in both proteins. The 8-mercapto-FAD peroxidase yields an EH2 spectrum on reduction virtually identical to that seen with 8-mercapto-FAD glutathione reductase, but no distinct EH2.NADH form appears. The dramatic decreases in reactivity at the flavin 2- and 4-positions for both the peroxidase and the oxidase, assessed with the reconstituted 2- and 4-thio-FAD enzymes, suggest that these positions are buried by elements of both protein structures. Furthermore, reconstitution of the peroxidase with the higher potential 2- and 4-thioflavins yields enzyme forms which are fully reducible with 1.4 eq of NADH/FAD, giving rise to stable thio-FADH2.NAD+ complexes. This behavior closely mimics that of the native NADH oxidase and provides further evidence supporting the hypothesis that a major functional distinction between the two structurally related proteins is determined by the redox potential and/or NADH reactivity of the bound flavin coenzyme.     

Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase

5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD. Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD. The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1). Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate. The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins. Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state. Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%). The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme. Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence. On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox. In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely. Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox. The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.