Glycine Oxidase from Bacillus subtilis
2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês
10.1074/jbc.m111095200
ISSN1083-351X
AutoresViviana Job, Giorgia Letizia Marcone, Mirella S. Pilone, Loredano Pollegioni,
Tópico(s)Polyamine Metabolism and Applications
ResumoGlycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) andd-amino acids (d-alanine,d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The ∼300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K m value ford-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis. Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) andd-amino acids (d-alanine,d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The ∼300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K m value ford-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis. Glycine oxidase (GO) 1GOglycine oxidaseDAAOd-amino acid oxidaseSOXsarcosine oxidaseSDHsarcosine dehydrogenaseDMGDHdimethylglycine dehydrogenaseMSOXmonomeric sarcosine oxidasePIPOXpipecolate oxidaseDASPOd-aspartate oxidaseMTOXN-methyltryptophan oxidaseTSOXheterotetrameric sarcosine oxidaseEFloxoxidized enzymeEFlseqenzyme flavin semiquinoneEFlredreduced enzymeAPP4-aminoantipyrine is a new flavoenzyme that was discovered in 1997 following the complete sequencing of the Bacillus subtilis genome (1Kunst F. Ogasawara N. Moszer I. Albertini A.M. Alloni G. Azevedo V. Bertero M.G. et al.Nature. 1997; 390: 249-256Crossref PubMed Scopus (3157) Google Scholar). Two previous investigations reported on the expression of the yjbR gene product (up to 3.9% of total soluble proteins in crude extract) and on its purification and partial characterization in Escherichia coli (2Nishiya Y. Imanaka T. FEBS Lett. 1998; 438: 263-266Crossref PubMed Scopus (62) Google Scholar). 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. The purified protein is a homotetrameric flavoenzyme that catalyzes the oxidation of various amines (e.g. sarcosine, N-ethylglycine and glycine) and d-amino acids (e.g. d-alanine, d-proline, d-valine, etc.). GO seems to partially share substrate specificity with various flavooxidases, such as d-amino-acid oxidase (DAAO) (EC1.4.3.3) and sarcosine oxidase (SOX), and also appears to be stereospecific in oxidizing the d-isomer of the amino acids tested. 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. glycine oxidase d-amino acid oxidase sarcosine oxidase sarcosine dehydrogenase dimethylglycine dehydrogenase monomeric sarcosine oxidase pipecolate oxidase d-aspartate oxidase N-methyltryptophan oxidase heterotetrameric sarcosine oxidase oxidized enzyme enzyme flavin semiquinone reduced enzyme 4-aminoantipyrine GO exhibits highest sequence homology with the β subunit of TSOX (EC1.5.3.1), sarcosine dehydrogenase (SDH) (EC 1.5.99.1) and dimethylglycine dehydrogenase (DMGDH) (EC 1.5.99.2) (24–27% identity), in particular with the N-terminal regions corresponding to the flavin-binding domain. Only a modest similarity is observed with the sequences of monomeric sarcosine oxidase (MSOX) (EC 1.5.3.1), pipecolate oxidase (PIPOX) (EC 1.5.99.3) and DAAO ord-aspartate oxidase (DASPO) (EC 1.4.3.1) (18–21% identity). 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. DAAO (containing 1 mole of non-covalently bound FAD per 40 kDa monomer) catalyzes the oxidative deamination of neutral and (with a lower efficiency) basic d-amino acids to give the corresponding α-keto acids, ammonia and hydrogen peroxide (4Pilone M.S. Cell. Mol. Life Sci. 2000; 57: 1732-1747Crossref PubMed Scopus (187) Google Scholar). Acidic d-amino acids are oxidized by DASPO.N-Methyltryptophan oxidase (MTOX) (EC 1.5.3.1) is a monomeric flavoenzyme (42 kDa) from E. coli containing FAD covalently linked to a cysteine residue (5Wagner M.A. Kanna P. Schuman Jorns M. Biochemistry. 1999; 38: 5588-5595Crossref PubMed Scopus (69) Google Scholar). PIPOX is a mammalian enzyme similar in size to MTOX and to the β subunit of MSOX (44 kDa) and also contains a single covalently bound flavin (6Mihalik S., J. McGuinnes M. Watkins P.A. J. Biol. Chem. 1991; 266: 4822-4830Abstract Full Text PDF PubMed Google Scholar). Sarcosine oxidases have been isolated from several bacteria and belong to two different classes of enzymes: heterotetrameric (TSOX) and monomeric (MSOX) enzymes. All bacterial SOXs catalyze the oxidative demethylation of sarcosine to yield glycine, hydrogen peroxide, and formaldehyde (7Wagner M.A. Schuman Jorns M. Arch. Biochem. Biophys. 1997; 342: 176-181Crossref PubMed Scopus (27) Google Scholar). The heterotetrameric SOXs contain four different subunits (from 10 to 100 kDa) and also contain non-covalently bound FAD, non-covalently bound NAD+, and covalently bound FMN, which is linked to the β subunit (42–45 kDa). The monomeric SOXs are similar in size to the β subunit of TSOX and contain covalently bound FAD. Only TSOXs use tetrahydrofolate as a substrate, and, in this regard, they resemble mammalian SDH and DMGDH (7Wagner M.A. Schuman Jorns M. Arch. Biochem. Biophys. 1997; 342: 176-181Crossref PubMed Scopus (27) Google Scholar). In mammals, these two enzymes catalyze the oxidative demethylation of sarcosine in the mitochondria. They are monomeric enzymes (97 and 96 kDa, respectively) containing a single, covalently bound flavin and are considered to be the two main folate-containing enzymes in rat liver mitochondria. Furthermore, these enzymes are linked to the electron transport chain and form 5,10-methylenetetrahydrofolate (8Cook R.J. Misono K.S. Wagner C. J. Biol. Chem. 1985; 260: 12998-13002Abstract Full Text PDF PubMed Google Scholar). This paper focuses on characterizing the substrate-binding site and the properties of the prosthetic group in recombinant GO. We identify the products of the reaction catalyzed by GO on a number of substrates, as well as compounds that act as inhibitors and which have proved useful in probing the reaction mechanism of the enzyme. The main goal of this project is to elucidate the structure-function relationships in GO, with the ultimate aim of clarifying the modulation of the substrate specificity in enzymes active on similar compounds. Horseradish peroxidase, formaldehyde dehydrogenase from Pseudomonas putida, monomeric sarcosine oxidase fromB. subtilis, tetrameric sarcosine oxidase fromCorynebacterium sp., d-amino acids, xanthine, xanthine oxidase, ampicillin, chloramphenicol, and all other compounds were purchased from Sigma. Glutamate dehydrogenase from bovine liver was from Roche Diagnostics. 5-Deazaflavin was a generous gift of Dr. Sandro Ghisla (University of Konstanz, Konstanz, Germany). Kinetic experiments were performed at 25 °C in 75 mmsodium pyrophosphate and 5 μm FAD, pH 8.5, the other experiments at 15 °C in 50 mm sodium pyrophosphate, pH 8.5, or in 50 mm potassium phosphate, pH 7.0, containing 10% glycerol, unless stated otherwise. The pT7-HisGO expression plasmid 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. was transferred, for protein production, to the host BL21(DE3)pLysS E. coli strains. Recombinant E. coli cells were grown at 37 °C, and GO expression was induced by adding isopropyl-1-thio-β-d-galactopyranoside as reported. 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. GO was purified from the crude extract by chromatography on a HiTrap Chelating affinity column using anÄKTA FPLC system (Amersham Biosciences, Inc.). The fractions containing GO activity were pooled and loaded on a PD10 column equilibrated with 50 mm sodium pyrophosphate buffer, pH 8.5, containing 10% glycerol. Thus, the recombinant enzyme used in these experiments contains an N-terminal His tag sequence. Glycine oxidase activity was assayed using three different methods: i) polarographically using an oxygen electrode at 25 °C and at air saturation ([O2] = 0.253 mm) 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication.; and ii and iii) spectrophotometrically via determination of H2O2 with an enzyme-coupled assay using horseradish peroxidase (6–10 units/ml) ando-dianisidine (0.32 mg/ml), assuming Δε440 = 13,000 m−1 cm−1 (9Chlumsky L.J. Zhang L. Ramsey A.J. Schuman Jorns M. Biochemistry. 1993; 32: 11132-11142Crossref PubMed Scopus (24) Google Scholar), or 2 mm phenol and 1.5 mm 4-aminoantipyrine (10Mori N. Sano Y. Tani Y. Yamada H. Agric. Biol. Chem. 1980; 44: 1391-1397Crossref Scopus (13) Google Scholar), assuming Δε505 = 6580 m−1cm−1. All assays were performed on a final volume of 1 ml in 75 mm sodium pyrophosphate buffer, pH 8.5, using 10 mm sarcosine as the substrate. The polarographic assay solution also contained 5 μm FAD. One GO unit is defined as the amount of enzyme that converts 1 μmol of substrate (sarcosine or oxygen) or that produces 1 μmol of hydrogen peroxide per minute at 25 °C. Photoreduction in the presence of EDTA was conducted as described in Ref. 11Massey V. Hemmerich P. Biochemistry. 1978; 17: 9-16Crossref PubMed Scopus (301) Google Scholar, using an anaerobic cuvette, made anaerobic by alternative cycles of vacuum and O2-free argon, containing 12 μm enzyme, 5 mm EDTA, and 0.5 μm 5-deazaflavin. The cuvette with the protein solution was kept in a water bath at 10 °C and ∼7 cm from a 150-W quartz halogen light source. The cuvette was removed at specified time intervals, and the progress of the reaction was followed spectrophotometrically (11Massey V. Hemmerich P. Biochemistry. 1978; 17: 9-16Crossref PubMed Scopus (301) Google Scholar). The thermodynamic stability of the semiquinone was determined by adding 5 μm benzyl viologen from a side arm of the cuvette after the photoreduction was complete. The disproportionation of the semiquinone was then followed until equilibration was reached (for up to 24 h at 4 °C) (12Sander S.A. Williams C.H. Massey V. J. Biol. Chem. 1999; 274: 22289-22295Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Flavin and protein fluorescence measurements were carried out in a Jasco FP750 spectrofluorometer at pH 7.0 and 15 °C. Protein fluorescence emission was monitored at an excitation of 280 nm, and flavin fluorescence emission at an excitation of 450 nm, using an excitation slit = 5 nm and an emission slit = 10 nm. Flavin was extracted from GO upon unfolding by heating the enzyme at 95 °C for 5 min and removing denatured protein by centrifugation. The flavin in the supernatant was identified by its absorption spectrum as compared with that of native FAD and by fluorescence spectroscopy before and after treatment with phosphodiesterase, which generates FMN and increases the fluorescence yield (13Whitby L.G. Biochem. J. 1953; 54: 437-442Crossref PubMed Scopus (314) Google Scholar). The extracted flavin was analyzed by chromatography on an Aquapore RP-300 column, according to the method described in Ref. 14Light D.R. Walsh C. Marletta A. Anal. Biochem. 1980; 109: 87-93Crossref PubMed Scopus (77) Google Scholar. Peak detection was monitored following the absorbance at 450 nm, and the fluorescence at 530 nm (excitation at 450 nm). The peaks were identified by comparing them with the retention time of chromatographically pure flavins. Redox potentials for the EFlox/EFlseq, and EFlseq/EFlred couples of GO were determined by dye equilibration (15Minnaert K. Biochim. Biophys. Acta. 1965; 110: 42-56Crossref PubMed Google Scholar), using the xanthine/xanthine oxidase reduction system at pH 7.0 and 15 °C (16Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins. Walter de Gruyter and Co., Berlin1991: 59-66Google Scholar). The reaction was initiated by adding 10–30 nm xanthine oxidase to an anaerobic cuvette containing ∼10 μm enzyme, 0.2 mm xanthine, 5 μm methyl viologen, and 5–10 μm of the appropriate dye (16Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins. Walter de Gruyter and Co., Berlin1991: 59-66Google Scholar). The amount of oxidized and reduced dye was determined at a wavelength at which the enzyme shows no absorbance (>550 nm), and the amount of oxidized and reduced enzyme was determined either at an isosbestic point for the dye or by subtracting the dye in the 400–470-nm region. Data were analyzed as described in Ref. 15Minnaert K. Biochim. Biophys. Acta. 1965; 110: 42-56Crossref PubMed Google Scholar. Dissociation constants for the ligands were measured spectrophotometrically by adding small volumes (1–10 μl) of concentrated stock solutions to samples containing 800 μl of ∼10 μm enzyme at 15 °C. The change in absorbance at the wavelengths at which the modification was more pronounced was plotted as a function of ligand concentration, after correction for any volume change; K d values were determined according to Ref. 17Strickland S. Palmer G. Massey V. J. Biol. Chem. 1975; 250: 4048-4052Abstract Full Text PDF PubMed Google Scholar. For reactions with sulfite, the reagent was prepared just before use as a 500-mm stock solution in 50 mm potassium phosphate, pH 7.0, or in 50 mm sodium pyrophosphate, pH 8.5, containing 10% glycerol, and aliquots were then added to enzyme solution at 15 °C. The rate of decay (k off, dissociation of SO32−) for the N-(5)-sulfite adduct was kinetically determined spectrophotometrically after removing excess sulfite by gel filtration (application of 1-ml samples to a Sephadex G-25 column, void volume 2.5 ml, at 15 °C). The GO-sulfite reaction was investigated by means of stopped-flow spectrophotometric measurements. The experiments were performed at 25 °C in a BioLogic SFM-300 stopped-flow spectrophotometer equipped with a thermostat, a cell with a path length of 1 cm, and with a J&M diode array detector. The reactions were routinely recorded in the 250–700-nm wavelength range using the buffer already described but containing 2% glycerol (final concentration). Reaction rates were calculated by extracting traces at 456 nm and fitting them to a sum of exponential equations using Kaleidagraph. To study the reaction catalyzed by GO, the products formed during the reaction with 100 mmd-alanine, 10 mm glycine, 50 mm N-ethylglycine, and 10 mm sarcosine were analyzed using different methods. α-Keto acid production was estimated as the production of 2,4-dinitrophenylhydrazone derivatives by a reaction with 2,4-dinitrophenylhydrazine (18Nagata Y. Shimojo T. Akino T. Int. J. Biochem. 1988; 20: 1235-1238Crossref PubMed Scopus (25) Google Scholar). After the addition of GO to a mixture containing the substrate in 75 mm sodium pyrophosphate, pH 8.5, aliquots (300 μl) were withdrawn at different times and mixed with (150 μl) 1 mm2,4-dinitrophenylhydrazine (dissolved in 1 n HCl). After incubation at 37 °C for 10 min, 1.05 ml of 0.6 n NaOH was added; the absorbance at 445 nm was measured after incubation for another 5 min at room temperature. Calibration curves were achieved using both glyoxylate and pyruvate, since the corresponding α-keto acid derivatives show different absorbance spectra and extinction coefficients. The ammonia produced during the reaction of GO was determined using a coupled assay with glutamate dehydrogenase from beef, according to the manufacturer's instructions and following the oxidation of NADH at 340 nm. GO was added to the assay mixture (1 ml) containing the substrate, 5 mm 2-oxoglutarate, 0.25 mm NADH, and 20 units of glutamate dehydrogenase in 65 mm sodium pyrophosphate, pH 8.5. A calibration curve was achieved using known amounts of ammonium chloride. To assess the production of formaldehyde, the GO reaction was coupled to the formaldehyde dehydrogenase NAD-dependent reaction (fromP. putida) following the production of NADH at 340 nm. GO was added to a mixture reaction (350 μl) containing the substrate, 2 mm NAD+, and 200 μg of formaldehyde dehydrogenase in 65 mm sodium pyrophosphate, pH 8.5. A calibration curve was determined using formaldehyde; blank reactions contained all the assay components except GO. MSOX (B. subtilis) and TSOX (Corynebacterium sp.) were used as a positive control, and DAAO (Rhodotorula gracilis) as a negative control. H2O2 production was determined using the previously described coupled method with horseradish peroxidase. The scheme of the strategy devised to obtain cDNA coding for the chimeric His-tagged GO has been reported previously. 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. His-GO was expressed in E. coli BL21(DE3)pLysS cells transformed as a totally soluble protein: about 2.4 units/g wet weight (≈2 mg of GO/g of cell) of enzyme was expressed, the purification yield being ≈100% (>65 mg of pure GO from 6 liters of culture). 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. The recovered His-tagged GO is fully active since the addition of exogenous FAD or FMN to the assay mixture does not increase the GO activity (specific activity on sarcosine as the substrate is 1.06 units/mg protein at 25 °C). The final preparation was homogeneous at 95% (SDS-PAGE reveals the presence of a single polypeptide chain corresponding to an apparent molecular mass of ∼49.4 ± 1.1 kDa) and was stable when stored at 80 °C for several months. The spectrum of GO in the oxidized state is shown in Fig. 1 A, where it is compared with that of the same sample after heat denaturation and centrifugation (relevant spectral data are summarized in TableI). This latter spectrum is identical to that of free FAD. The protein pellet is colorless, and, after resolubilization using 6 m guanidinium chloride, no absorbance bands are evident in the visible portion of the spectrum, indicating that the flavin cofactor is not covalently bound to the protein moiety. High pressure liquid chromatography of the extracted flavin, using pure FAD and FMN as internal standards (R t = 4.12 and 4.32 min, respectively), showed that all the extracted flavin was present as FAD (single peak with aR t = 4.14 min). This result is in agreement with a 7-fold increase in flavin fluorescence and with the slower chromatographic mobility (R t = 4.33 min) that was observed when phosphodiesterase (0.5 μg/μl) was added to the extracted flavin, as expected due to the conversion of the FAD molecule to FMN. Calculations using the absorption coefficient for free FAD of 11.3 mm−1 cm−1 resulted in an estimated flavin absorption coefficient of 11.8 mm−1 cm−1 for the holoenzyme and an A 274/A 456 ratio of ∼8.6. According to the amount of FAD extracted and the concentration of the protein in the pellet after heat denaturation (determined from the absorbance at 280 nm of the protein re-dissolved in 6 mguanidinium chloride and using the extinction coefficient theoretically calculated from the amino acid sequence, ε = 55190m−1 cm−1), a ratio of 1 mole of FAD:1 mole of enzyme monomer was determined. The 450-nm band exhibited a shoulder of ∼470 nm that was similar to the resolved 450-nm band observed for free flavins in non-polar solvents (19Harbury H.A. LaNoue K.F. Loach P.A. Amick R. Proc. Natl. Acad. Sci. U. S. A. 1959; 45: 1708-1717Crossref PubMed Google Scholar) and similar to the spectrum of MSOX (20Willie A. Edmondson D.E. Schuman Jorns M. Biochemistry. 1996; 35: 5292-5299Crossref PubMed Scopus (34) Google Scholar) and R. gracilis DAAO (21Pilone Simonetta M. Pollegioni L. Casalin P. Curti B. Ronchi S. Eur. J. Biochem. 1989; 180: 199-204Crossref PubMed Scopus (63) Google Scholar).Table ISpectral properties of glycine oxidase from B. subtilisRedox stateValueEFlox, λmax(nm)273, 379, 456 ε (mM−1cm−1)103.2, 9.51, 11.81 Absorbance ratios8.7, 10.9, 1 Fluorescence emission (λmax, nm) (λexc = 280; 450 nm)342, 525 % of that of free FAD16.8EFlred, λmax(nm)267, 346 ε (mM−1cm−1)110, 6.5 Fluorescence emission (λmax, nm) (λexc = 280; 340 nm)343, 422EFlseq, λmax (nm)268, 371, 477 ε (mM−1 cm−1)113.6, 15.8, 5.3 Open table in a new tab GO shows a marked dependence of the absorption spectrum of the oxidized form on the pH value. When the pH value of a solution is shifted from 8.0 to high values (>11) a hypochromic shift is observed for the near-UV band (from 376 to 350 nm), as well as a bathochromic shift of the 455-nm peak (data not shown). The spectral changes resemble those observed with free FAD upon ionization of the N(3)-H position (22Massey V. Ganther H. Biochemistry. 1965; 4: 1161-1173Crossref PubMed Scopus (180) Google Scholar). This is consistent with N(3)-H of bound FAD not being ionized at physiological pH. The pK a value of this flavin position was determined by plotting the absorbance at 385 nm against pH; it is increased compared with free FAD (10.6 ± 0.1versus ∼10.4) (22Massey V. Ganther H. Biochemistry. 1965; 4: 1161-1173Crossref PubMed Scopus (180) Google Scholar). GO undergoes denaturation at high pH values; it is fully denatured when brought to pH 12 and then changing the buffer by desalting on a PD10 column and bringing the pH to 7. Worthy of note is that a change in the absorbance spectrum is also evident when the pH value is shifted from 8.0 to a more acidic value. Unluckily, due to the low stability of GO at pH ≤6.5, 2V. Job, G. Molla, M. S. Pilone, and L. Pollegioni, submitted for publication. this ionization can not be followed over a pH range that is wide enough to determine the corresponding pK a . The flavin fluorescence emission of GO is rather weak in the oxidized state with λmax(emiss) ≈525 nm (Table I). Compared with flavin standards the relative fluorescence quantum yield of protein-bound FAD is less than 17% of free FAD. Typically, the anionic semiquinone stabilizes in this class of flavoprotein oxidases (23Massey V. Hemmerich P. Biochem. Soc. Trans. 1980; 8: 246-257Crossref PubMed Scopus (289) Google Scholar). GO is photoreduced in the presence of EDTA and 5-deazaflavin at 15 °C and pH 8.5 forming a red anionic semiquinone. Three isosbestic points are observed at 340, 411, and 506 nm while the semiquinone is being formed (Fig.2). The amount of semiquinone form stabilized by GO at pH 7.0 and 8.5 (as determined by anaerobic photoreduction (11Massey V. Hemmerich P. Biochemistry. 1978; 17: 9-16Crossref PubMed Scopus (301) Google Scholar) until the spectrum of the flavin semiquinone (EFlseq) reached a maximum) represents near-complete formation of EFlseq (≈95%). When oxygen is added, essentially, complete re-oxidation is observed. The anionic semiquinone species of GO is fully stable when kept in the dark but slowly disproportionates to the oxidized and reduced forms upon anaerobic addition of benzyl viologen, the end-product containing the final amount of thermodynamically stabilized semiquinone (11Massey V. Hemmerich P. Biochemistry. 1978; 17: 9-16Crossref PubMed Scopus (301) Google Scholar). After 24 h, when equilibrium was finally reached, ∼40 and 24% of the photoreduced GO remained in the semiquinone form at pH 7.0 and 8.5, respectively. These results are compatible with a kinetic stabilization of the (red) anionic flavin semiquinone as also observed with other flavoprotein oxidases (23Massey V. Hemmerich P. Biochem. Soc. Trans. 1980; 8: 246-257Crossref PubMed Scopus (289) Google Scholar). Anaerobic addition of an excess of glycine resulted in instantaneous flavin enzyme reduction, yielding a spectrum like that of the reduced flavin, with a maximum at 346 nm (line 3 in Fig. 1 A). The anaerobic reduction demonstrates that GO is competent in catalysis. An anaerobic titration of GO with glycine is depicted in Fig. 1 B. The intercept of the initial slope of the titration curve with the maximal observed change at 455 nm (inset of Fig. 1 B) indicates a stoichiometry ∼1 for the reaction with the substrate (19.0 nmol of enzyme and 19.5 nmol of substrate). In the reduced state, the fluorescence emission at 530 nm (excitation at 450 nm) is essentially lost. The ability to bind sulfite and to form reversible covalent N(5) adducts distinguishes oxidases from other classes of flavoproteins (23Massey V. Hemmerich P. Biochem. Soc. Trans. 1980; 8: 246-257Crossref PubMed Scopus (289) Google Scholar, 24Massey V. Müller F. Feldberg R. Schuman M. Sullivan P.A. Howell L.G. Mayhew S.G. Matthews R.G. Foust G.P. J. Biol. Chem. 1969; 244: 3999-4006Abstract Full Text PDF PubMed Google Scholar). It is generally assumed that in this class of flavoenzymes a positive (partial) charge near the flavin N(1)-C(2)=O locus inductively promotes the process (23Massey V. Hemmerich P. Biochem. Soc. Trans. 1980; 8: 246-257Crossref PubMed Scopus (289) Google Scholar). GO reversibly forms flavin N(5)-sulfite adducts, which essentially bleaches the oxidized flavin spectrum (Fig.3 A). The GO complex is quite stable; the K d values obtained for GO at pH 7.0 and 8.5 (13 and 41 μm, see inset of Fig.3 A) are in the micromolar range, analogously to many oxidases (24Massey V. Müller F. Feldberg R. Schuman M. Sullivan P.A. Howell L.G. Mayhew S.G. Matthews R.G. Foust G.P. J. Biol. Chem. 1969; 244: 3999-4006Abstract Full Text PDF PubMed Google Scholar). The reversibility of sulfite binding was assessed by measuring the time-dependent recovery of the flavin absorbance spectrum of the oxidized enzyme upon filtration over Sephadex G-25 (k off ≈0.04 s−1 at pH 8.5). At pH 7.0, the reaction is very fast, and thus the value determined (k off ≥ 0.1 s−1) gives the lower limit. The reaction with sulfite was thus followed by means of stopped-flow spectroscopy by mixing a fixed amount of GO with different concentrations of sulfite. As shown in Fig. 3 B, with GO the adduct formation depends monophasically on the sulfite concentration and yields a spectrum that is typical of the flavin N(5)-sulfite adduct only when a high, saturating concentration of sulfite is used (Fig. 3) (24Massey V. Müller F. Feldberg R. Schuman M. Sullivan P.A. Howell L.G. Mayhew S.G. Matthews R.G. Foust G.P. J. Biol. Chem. 1969; 244: 3999-4006Abstract Full Text PDF PubMed Google Scholar). Binding is a second-order process, and the plot ofk obs versus sulfite concentration is linear (not shown); values for the rates of complex formation (k on = 1660 m−1s−1) and dissociation (k off = 0.20 s−1) were estimated from the slope andy-intercept, respectively. The latter value is compatible with that obtained by following the reappearance of the oxidized flavin spectrum upon removal of excess sulfite by gel filtration. TheK d value calculated for the GO complex based on the rate constants observed (K d =k off/k on = 0.12 mm) is in fairly good agreement with that (K d = 67 μm) determined from the 455 nm absorbance change as a function of sulfite concentration (Fig.3 B, inset). In contrast, a ∼10-fold difference between the K d values determined by the static spectrophotometric titration and the kinetic measurements is evident (this discrepancy may be ascribed to the 10 °C difference in temperature used in the two determinations). The method described in Ref. 16Massey V. Curti B. Ronchi S. Zanetti G. Flavins and Flavoproteins. Walter de Gruyter and Co., Berlin1991: 59-66Google Scholar was used to determine the midpoint redox potentials for the transfer of electrons to the flavin at 15 °C and pH 7.0. The separation of the two redox potentials was determined from the maximal percentage of the semiquinone reached during the reduction using methyl viologen (E m = −440 mV) as dye. When benzyl viologen was used instead of methyl viologen, the broad absorbance band at ∼600 nm, corresponding to the conversion of oxidized benzyl viologen to the reduced form, appeared before the semiquinone form of the enzyme was completely reduced (see below), i.e. the potential for transfer of the second electron to the enzyme is close to that of the dye. For this reason, all the subsequent experiments were carried out using methyl viologen as the final acceptor. Thus, when the xanthine oxidase-mediated reduction of GO was monitored in the absence of a reference dye, the percentage of semiquinone formed during the reduction is ≈95%, i.e. the potentials for transfer of
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