Spectroscopic and kinetic properties of recombinant choline oxidase from Arthrobacter globiformis

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, with molecular oxygen acting as primary electron acceptor. Recently, the recombinant enzyme expressed in Escherichia coli was purified to homogeneity and shown to contain FAD in a mixture of oxidized and anionic semiquinone redox states [Fan et al. (2003) Arch. Biochem. Biophys., in press]. In this study, methods have been devised to convert the enzyme-bound flavin semiquinone to oxidized FAD and vice versa, allowing characterization of the resulting forms of choline oxidase. The enzyme-bound oxidized flavin showed typical UV-vis absorbance peaks at 359 and 452 nm (with epsilon(452) = 11.4 M(-1) cm(-1)) and emitted light at 530 nm (with lambda(ex) at 452 nm). The affinity of the enzyme for sulfite was high (with a K(d) value of approximately 50 microM at pH 7 and 15 degrees C), suggesting the presence of a positive charge near the N(1)C(2)=O locus of the flavin. The enzyme-bound anionic flavin semiquinone was unusually insensitive to oxygen or ferricyanide at pH 8 and showed absorbance peaks at 372 and 495 nm (with epsilon(372) = 19.95 M(-1) cm(-1)), maximal fluorescence emission at 454 nm (with lambda(ex) at 372 nm), circular dichroic signals at 370 and 406 nm, and an ESR peak-to-peak line width of 13.9 G. Both UV-vis absorbance studies on the enzyme under turnover with choline and steady-state kinetic data with either choline or betaine aldehyde were consistent with the flavin semiquinone being not involved in catalysis. The pH dependence of the kinetic parameters at varying concentrations of both choline and oxygen indicated that a catalytic base is required for choline oxidation but not for oxygen reduction and that the order of the kinetic steps involving substrate binding and product release is not affected by pH.     

A hydrogen peroxide-forming NADH oxidase that functions as an alkyl hydroperoxide reductase in Amphibacillus xylanus

The Amphibacillus xylanus NADH oxidase, which catalyzes the reduction of oxygen to hydrogen peroxide with beta-NADH, can also reduce hydrogen peroxide to water in the presence of free flavin adenine dinucleotide (FAD) or the small disulfide-containing Salmonella enterica AhpC protein. The enzyme has two disulfide bonds, Cys128-Cys131 and Cys337-Cys340, which can act as redox centers in addition to the enzyme-bound FAD (K. Ohnishi, Y. Niimura, M. Hidaka, H. Masaki, H. Suzuki, T. Uozumi, and T. Nishino, J. Biol. Chem. 270:5812-5817, 1995). The NADH-FAD reductase activity was directly dependent on the FAD concentration, with a second-order rate constant of approximately 2.0 x 10(6) M(-1) s(-1). Rapid-reaction studies showed that the reduction of free flavin occurred through enzyme-bound FAD, which was reduced by NADH. The peroxidase activity of NADH oxidase in the presence of FAD resulted from reduction of peroxide by free FADH(2) reduced via enzyme-bound FAD. This peroxidase activity was markedly decreased in the presence of oxygen, since the free FADH(2) is easily oxidized by oxygen, indicating that this enzyme system is unlikely to be functional in aerobic growing cells. The A. xylanus ahpC gene was cloned and overexpressed in Escherichia coli. When the NADH oxidase was coupled with A. xylanus AhpC, the peroxidase activity was not inhibited by oxygen. The V(max) values for hydrogen peroxide and cumene hydroperoxide reduction were both approximately 150 s(-1). The K(m) values for hydrogen peroxide and cumene hydroperoxide were too low to allow accurate determination of their values. Both AhpC and NADH oxidase were induced under aerobic conditions, a clear indication that these proteins are involved in the removal of peroxides under aerobic growing conditions.     

Studies on the structure and mechanism of Streptococcus faecium L-alpha-glycerophosphate oxidase

An FAD-containing L-alpha-glycerophosphate oxidase has been purified to homogeneity from Streptococcus faecium. The purified protein exists as a dimer (subunit Mr = 65,000); each subunit contains 1 mol of FAD. The enzyme contains no iron, as determined by atomic absorption spectroscopy. The alpha-glycerophosphate oxidase reacts reversibly with sulfite to form a covalent N(5) adduct; it preferentially binds the anionic form of the native oxidized FAD, and it also stabilizes the p-quinonoid form of 8-mercapto-FAD. The enzyme shows an unusually high reactivity with ferricyanide in the absence of oxygen; however, there is no evidence for any superoxide ion (O2-.) generation under standard assay conditions. Dithionite titrations of the enzyme reveal an unusual pH dependence for the stabilization of the flavin semiquinone; only at pH 8.5 does significant anionic semiquinone accumulate. L-alpha-Glycerophosphate rapidly reduces the enzyme-bound FAD; in addition, a small amount of catalytically insignificant red semiquinone appears under these conditions. The 5-deaza-FAD-reconstituted enzyme is also reduced by substrate, strongly suggesting that a radical mechanism is not involved in the oxidation of alpha-glycerophosphate. Furthermore, nitroethane anion reduces the native enzyme; this observation suggests that an electron transfer mechanism involving a substrate carbanion is possible with this enzyme.     

15N- and 13C-NMR investigations of glucose oxidase from Aspergillus niger

The apoprotein of glucose oxidase from Aspergillus niger was reconstituted with specifically 15N- and 13C-enriched FAD derivatives and investigated by 15N- and 13C-NMR spectroscopy. On the basis of the 15N-NMR results it is suggested that, in the oxidized state of glucose oxidase, hydrogen bonds are formed to the N(3) and N(5) positions of the isoalloxazine system. The hydrogen bond to N(3) is more pronounced than that to N(5) as compared with the respective hydrogen bonds formed between FMN and water. The resonance position of N(10) indicates a small decrease in sp2 hybridization compared to free flavin in water. Apparently the isoalloxazine ring is not planar at this position in glucose oxidase. Additional hydrogen bonds at the carbonyl groups of the oxidized enzyme-bound FAD were derived from the 13C-NMR results. A strong downfield shift observed for the C(4a) resonance may be ascribed in part to the decrease in sp2 hybridization at the N(10) position and to the polarization of the carbonyl groups at C(2) and C(4). The polarization of the isoalloxazine ring in glucose oxidase is more similar to FMN in water than to that of tetraacetyl-riboflavin in apolar solvents. In the reduced enzyme the N(1) position is anionic at pH 5.6. The pKa is shifted to lower pH values by at least 1 owing to the interaction of the FAD with the apoprotein. As in the oxidized state of the enzyme, a hydrogen bond is also formed at the N(3) position of the reduced flavin. The N(5) and N(10) resonances of the enzyme-bound reduced FAD indicate a decrease in the sp2 character of these atoms as compared with that of reduced FMN in aqueous solution. Some of the 15N- and 13C-resonance positions of the enzyme-bound reduced cofactor are markedly pH-dependent. The pH dependence of the N(5) and C(10a) resonances indicates a decrease in sp2 hybridization of the N(5) atom with increasing pH of the enzyme solution.     

Purification and characterization of an aldehyde oxidase from Pseudomonas sp. KY 4690

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O(2) as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5'-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum-molybdpterin-cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe-2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.     

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

A highly active O₂-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l-amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O₂-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l-amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl₂ on the two examined reactions could also be demonstrated with this O₂-evolving Photosystem II complex: CaCl₂ stimulated photosynthetic O₂ evolution, but inhibited the l-amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l-amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l-amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.     

Reactivity of chicken liver xanthine dehydrogenase containing modified flavins

Native FAD was removed from chicken liver xanthine dehydrogenase (XDH) and replaced with a number of artificial flavins of different redox potential. Dithionite titration of the 2-thio-FAD- or 4-thio-FAD (high potential)-containing enzymes showed that the first center to be reduced was the flavin. With native enzyme, iron-sulfur centers are the first to be reduced. With the low potential flavin, 6-OH-FAD, the enzyme-bound flavin was the last center to be reduced in reductive titration with xanthine. These shifts in the reduction profile support the hypothesis that the distribution of reducing equivalents in multi-center oxidation-reduction enzymes of this type is determined by the relative potentials of the centers. The reaction of molecular oxygen with fully reduced 2-thio-FAD XDH or 4-thio-FAD XDH resulted in 5 electron eq being released in a fast phase and one in a slow phase. Reduction of these enzymes by xanthine was limited at a rate comparable to that for the release of urate from native XDH. Xanthine/O2 turnover with these enzymes (and native XDH) resulted in approximately 40-50% of the xanthine reducing equivalents appearing as superoxide. Steady state turnover experiments involving all modified flavin-containing enzymes, as well as native enzyme, showed that shifting the flavin potential either positive or negative relative to FAD caused a decrease in catalytic activity in the xanthine/NAD reductase reaction. In the case of the xanthine/O2 reductase activity, there is no simple obvious relationship between the activity and the redox potential of the reconstituted flavin.     

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.     

Electron transfer in acetohydroxy acid synthase as a side reaction of catalysis. Implications for the reactivity and partitioning of the carbanion/enamine form of (alpha-hydroxyethyl)thiamin diphosphate in a "nonredox" flavoenzyme

Acetohydroxy acid synthases (AHAS) are thiamin diphosphate- (ThDP-) and FAD-dependent enzymes that catalyze the first common step of branched-chain amino acid biosynthesis in plants, bacteria, and fungi. Although the flavin cofactor is not chemically involved in the physiological reaction of AHAS, it has been shown to be essential for the structural integrity and activity of the enzyme. Here, we report that the enzyme-bound FAD in AHAS is reduced in the course of catalysis in a side reaction. The reduction of the enzyme-bound flavin during turnover of different substrates under aerobic and anaerobic conditions was characterized by stopped-flow kinetics using the intrinsic FAD absorbance. Reduction of enzyme-bound FAD proceeds with a net rate constant of k' = 0.2 s(-1) in the presence of oxygen and approximately 1 s(-1) under anaerobic conditions. No transient flavin radicals are detectable during the reduction process while time-resolved absorbance spectra are recorded. Reconstitution of the binary enzyme-FAD complex with the chemically synthesized intermediate 2-(hydroxyethyl)-ThDP also results in a reduction of the flavin. These data provide evidence for the first time that the key catalytic intermediate 2-(hydroxyethyl)-ThDP in the carbanionic/enamine form is not only subject to covalent addition of 2-keto acids and an oxygenase side reaction but also transfers electrons to the adjacent FAD in an intramolecular redox reaction yielding 2-acetyl-ThDP and reduced FAD. The detection of the electron transfer supports the idea of a common ancestor of acetohydroxy acid synthase and pyruvate oxidase, a homologous ThDP- and FAD-dependent enzyme that, in contrast to AHASs, catalyzes a reaction that relies on intercofactor electron transfer.     

Substrate orientation in xanthine oxidase: crystal structure of enzyme in reaction with 2-hydroxy-6-methylpurine

Xanthine oxidoreductase catalyzes the final two steps of purine catabolism and is involved in a variety of pathological states ranging from hyperuricemia to ischemia-reperfusion injury. The human enzyme is expressed primarily in its dehydrogenase form utilizing NAD+ as the final electron acceptor from the enzyme's flavin site but can exist as an oxidase that utilizes O2 for this purpose. Central to an understanding of the enzyme's function is knowledge of purine substrate orientation in the enzyme's molybdenum-containing active site. We report here the crystal structure of xanthine oxidase, trapped at the stage of a critical intermediate in the course of reaction with the slow substrate 2-hydroxy-6-methylpurine at 2.3A. This is the first crystal structure of a reaction intermediate with a purine substrate that is hydroxylated at its C8 position as is xanthine and confirms the structure predicted to occur in the course of the presently favored reaction mechanism. The structure also corroborates recent work suggesting that 2-hydroxy-6-methylpurine orients in the active site with its C2 carbonyl group interacting with Arg-880 and extends our hypothesis that xanthine binds opposite this orientation, with its C6 carbonyl positioned to interact with Arg-880 in stabilizing the MoV transition state.     

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.     

Reduced diphosphopyridine nucleotide peroxidase. Intermediates formed on reduction of the enzyme with dithionite or reduced diphosphopyridine nucleotide.

DPNH peroxidase is a flavin adenine dinucleotide-containing flavoprotein. Anaerobic titration of enzyme with dithionite has shown that the active site of the enzyme contains 2 mol of flavin and in addition 1 mol of a non-flavin electron acceptor that is tentatively identified as a disulfide group. Thus complete reduction of the enzyme requires 3 mol of dithionite per mole of active site. The first mole of dithionite reduces the non-flavin acceptor; complex formation between the reduced acceptor and one of the bound flavin molecules causes the formation of a long wavelength absorption band between 500 and 670 nm. The second mole of dithionite reduces the flavin that interacts with the reduced non-flavin group, and the long wavelength band disappears. The third mole of dithionite reduces the second mole of flavin. All groups are reoxidized in the presence of air. DPNH reacts with only two of the enzyme-bound electron acceptors. The first mole of DPNH reduces the non-flavin group to form an intermediate (I) that is almost identical with that formed by dithionite. The second mole of DPNH complexes with the second flavin of Intermediate I to form Intermediate II. This reaction causes a further absorbance increase in the long wavelength region; the tail of the absorption band now extends to 960 nm. The titration data (potassium phosphate, 0.05 M, pH 7.0) can be fitted with dissociation constants of 1 times 10-7 M for the formation of I, and 3 times 10-6 M for the conversion of I to II. In air, species II is oxidized to I; I is stable in air, but is oxidized stoichiometrically to oxidized enzyme by H2O2. Present evidence suggests that bound DPN-plus is responsible for the air stability of species I. Intermediate I, but not oxidized enzyme, reacts slowly with phenylmercuric acetate. This reaction causes loss of the air-stable intermediate and parallel loss in enzyme activity. The inactive enzyme cannot be reduced by DPNH to Species I; DPNH can, however, still react with the second flavin to form the autoxidizable complex. With other methods of enzyme inactivation there is also a direct correlation between residual enzyme activity and the ability of enzyme to form the air-stable intermediate. It is concluded that the air-stable intermediate is an important catalytic species.     

Differences in protein structure of xanthine dehydrogenase and xanthine oxidase revealed by reconstitution with flavin active site probes

The native flavin, FAD, was removed from chicken liver xanthine dehydrogenase and milk xanthine oxidase by incubation with CaCl2. The deflavoenzymes, still retaining their molybdopterin and iron-sulfur prosthetic groups, were reconstituted with a series of FAD derivatives containing chemically reactive or environmentally sensitive substituents in the isoalloxazine ring system. The reconstituted enzymes containing these artificial flavins were all catalytically active. With both the chicken liver dehydrogenase and the milk oxidase, the flavin 8-position was found to be freely accessible to solvent. The flavin 6-position was also freely accessible to solvent in milk xanthine oxidase, but was significantly less exposed to solvent in the chicken liver dehydrogenase. Pronounced differences in protein structure surrounding the bound flavin were indicated by the spectral properties of the two enzymes reconstituted with flavins containing ionizable -OH or -SH substituents at the flavin 6- or 8-positions. Milk xanthine oxidase either displayed no preference for binding of the neutral or anionic flavin (8-OH-FAD) or a slight preference for the anionic form of the flavin (6-hydroxy-FAD, 6-mercapto-FAD, and possibly 8-mercapto-FAD). On the other hand, the chicken liver dehydrogenase had a dramatic preference for binding the neutral (protonated) forms of all four flavins, perturbing the pK of the ionizable substituent greater than or equal to 4 pH units. These results imply the existence of a strong negative charge in the flavin binding site of the dehydrogenase, which is absent in the oxidase.     

Stimulation by D-glucose of mitochondrial oxidative events in islet cells.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.     

Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate

The catalytic efficiency (kcat/Km) of Escherichia coli flavin pyruvate oxidase can be stimulated 450-fold either by the addition of lipid activators or by limited proteolytic hydrolysis. Previous studies have shown that a functional lipid binding site is a mandatory prerequisite for the in vivo functioning of this enzyme (Grabau, C., and Cronan, J. E., Jr. (1986) Biochemistry 25, 3748-3751). The effect of activation on the transient state kinetics of partial reactions in the overall oxidative conversion of pyruvate to acetate and CO2 has now been examined. The rate of decarboxylation of pyruvate to form CO2 and hydroxyethylthiamin pyrophosphate for both activated and unactivated forms of the enzyme is identical within experimental error. The decarboxylation step was measured using substrate concentrations of the enzyme in the absence of an electron acceptor. The pseudo-first order rate constant for the decarboxylation step is 60-80 s-1. The rate of oxidation of hydroxyethylthiamin pyrophosphate and concomitant enzyme-bound flavin reduction was analyzed by stopped-flow methods utilizing synthetic hydroxyethylthiamin pyrophosphate. The pseudo-first order rate for this step with unactivated enzyme was 2.85 s-1 and increased 145-fold for lipid-activated enzyme to 413 s-1 and 61-fold for the proteolytically activated enzyme to 173 s-1. The analysis of a third reaction step, the reoxidation of enzyme-bound FADH, was also investigated by stopped-flow techniques utilizing ferricyanide as the electron acceptor. The rate of oxidation of enzyme.FADH is very fast for both unactivated (1041 s-1) and activated enzyme (645 s-1). The data indicate that the FAD reduction step is the rate-limiting step in the overall reaction for unactivated enzyme. Alternatively, the rate-limiting step in the overall reaction with the activated enzyme shifts to one of the partial steps in the decarboxylation reaction.     

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.     

Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.     

The role of the [2Fe-2s] cluster centers in xanthine oxidoreductase

Xanthine oxidoreductases (XOR), xanthine dehydrogenase (XDH, EC1.1.1.204) and xanthine oxidase (XO, EC1.2.3.2), are the best-studied molybdenum-containing iron-sulfur flavoproteins. The mammalian enzymes exist originally as the dehydrogenase form (XDH) but can be converted to the oxidase form (XO) either reversibly by oxidation of sulfhydryl residues of the protein molecule or irreversibly by proteolysis. The active form of the enzyme is a homodimer of molecular mass 290 kDa. Each subunit contains one molybdopterin group, two non-identical [2Fe-2S] centers, and one flavin adenine dinucleotide (FAD) cofactor. This review focuses mainly on the role of the two iron-sulfur centers in catalysis, as recently elucidated by means of X-ray crystal structure and site-directed mutagenesis studies. The arrangements of cofactors indicate that the two iron-sulfur centers provide an electron transfer pathway from molybdenum to FAD. However, kinetic and thermodynamic studies suggest that these two iron-sulfur centers have roles not only in the pathway of electron flow, but also as an electron sink to provide electrons to the FAD center so that the reactivity of FAD with the electron acceptor substrate might be thermodynamically controlled by way of one-electron-reduced or fully reduced state.     

Redox properties of microsomal reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase and cytochrome b5.

Hepatic NADH-cytochrome b5 reductase was reduced by 1 mol of dithionite or NADH per mol of enzyme-bound FAD, without forming a stable semiquinone or intermediate during the titrations. However, the addition of NAD+ to the partially reduced enzyme or illumination in the presence of both NAD+ and EDTA yielded a new intermediate. The intermediate had an absorption band at 375 nm and the optical spectrum resembled anionic semiquinones seen on reduction of other flavin enzymes. Electron paramagnetic resonance measurements confirmed the free-radical nature of the species. To explain the results, a disproportionation reaction between the oxidized and reduced NAD+ complexes (E-FAD-NAD+ + E-FADH2-NAD+ in equilibrium 2E-FADH.-NAD+) is assumed. Potentiometric titration of NADH-cytochrome b5 reductase at pH 7.0 with dithionite gave a midpoint potential of -258 mV; titration with NADH gave -160 mV. This difference may be due to a difference in the relative affinity of NAD+ for the reduced and oxidized forms of the enzyme. The effects of pH on the midpoint potential of the NAD+-free enzyme were very similar to those which have been measured with free FAD. At pH 7.0, midpoint potentials of trypsin-solubilized and detergent-solubilized cytochrome b5 were 13 and 0 mV, respectively.     

Analysis of interaction between the Arthrobacter sarcosine oxidase and the coenzyme flavin adenine dinucleotide by site-directed mutagenesis.

Sarcosine oxidase from Arthrobacter sp. TE1826 (SoxA) tightly binds with the coenzyme flavin adenine dinucleotide (FAD). The amino-terminal region of this enzyme was recognized as a part of the FAD-binding domain by homology search analysis. Comparison with other structurally well-known flavoproteins suggested that the aspartate residue at position 35 (D-35) and the motif sequence (six residues at positions 12 to 17) were important for the interaction with FAD. Site-directed mutagenesis of each position was performed, and mutant SoxAs were purified and characterized. When D-35 was substituted with glutamate, asparagine, and alanine, it was indicated that the carboxyl group of the side chain interacted with FAD. Changes in the enzyme-bound FAD were also observed from the altered spectral profiles. Thirteen mutant SoxAs were obtained by replacing amino acids in the motif sequence. Most of them showed inhibited or remarkably decreased sarcosine oxidase activity, and their spectral profiles were altered. However, some of them were reactivated by chloride ion. Their spectral profiles also became close to that of wild type in the presence of chloride ion. These results strongly suggest that the inhibition of interaction of enzyme with FAD was caused by the substitution in the motif and that it could be recovered under different conditions.