New flavins for old: artificial flavins as active site probes of flavoproteins.
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Affinity Labels↗
Biomedical subjects
V Massey
Publications and source records attributed to V Massey.
Electrophilic amination of a single methionine residue located at the active site of D-amino acid oxidase by O-(2,4-dinitrophenyl)hydroxylamine.
O-(2,4-Dinitrophenyl)hydroxylamine is a rapid active-site-directed inhibitor of D-amino acid oxidase: modification results in specific incorporation of an amine group into an accessible nucleophilic residue with concomitant release of 2,4-dinitrophenol. The reaction is prevented by the competitive inhibitor benzoate, indicating an active-site-directed reaction. A stoichiometry of 1-1.5 mol of amine residues per enzyme bound flavin adenine dinucleotide monomer was observed at pH 7.0. Amino acid and sequence analyses show that His-217 is not the target of the modification reaction. Dependence of the modification on pH, model studies on functional groups present on amino acids, and thiolysis studies on aminated enzyme collectively indicate that the modification is located on a methionine residue at or near the active site of the enzyme. Aminated enzyme, although spectrally similar to native enzyme, exhibits a 7-9-nm blue shift in the 455-nm flavin absorption. Benzoate perturbs the spectrum of aminated enzyme, but binding relative to native enzyme is much weaker (Kd ca. 300 times greater at pH 8.0).
Animals↗
Two-electron reduced mercuric reductase binds Hg(II) to the active site dithiol but does not catalyze Hg(II) reduction.
Mercuric reductase contains FAD and a redox-active disulfide which is reduced to a thiol/thiolate pair in two-electron reduced enzyme (EH2) (Fox, B. and Walsh, C.T. (1982) J. Biol. Chem. 257, 2498-2503). A charge transfer interaction between the thiolate and oxidized FAD gives EH2 a characteristic absorption spectrum, very similar to that found with other flavoprotein disulfide oxidoreductases. We have examined the reaction of EH2 with HgCl2 (+/- mercaptoethanol) in stopped-flow kinetic and static titration experiments. In the absence of mercaptoethanol, reaction of EH2 with HgCl2 yields a final spectrum which is indistinguishable from that of oxidized enzyme. The nature of the final species was examined by titration of enzyme thiols with 5,5'-dithiobis-2,2'-nitrobenzoic acid under denaturing conditions in the presence of NaI to displace any Hg(II) bound to enzyme thiols. These studies demonstrate that EH2 tightly complexes Hg(II) with its active site thiols, but is incapable of reducing Hg(II) to Hg0. For the latter reaction to occur, additional reducing equivalents are required. In catalysis, the enzyme must first be reduced to EH2 after which it cycles between EH2 and EH2 X NADPH forms. This is in contrast to other flavoprotein disulfide oxidoreductases which cycle between Eox and EH2 forms in catalysis (Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D., ed) 3rd Ed., Vol. 13, pp. 89-173, Academic Press, New York). With mercuric reductase, exogenous thiols are required for catalytic reduction of Hg(II) to Hg0. We have shown that this is due to prevention or reversal of formation of an abortive complex of Hg(II) with the thiol/thiolate pair of EH2.
Binding Sites↗
Preparation and some properties of 6-substituted flavins as active site probes for flavin enzymes.
6-Azidoflavins, 6-thiocyanatoflavins, and 6-mercaptoflavins at the lumiflavin, riboflavin, FMN, and FAD level were prepared from the corresponding 6-aminoflavins and some of their properties investigated. They are bound tightly by apoflavin enzymes which bind either riboflavin, FMN, or FAD. 6-Azidoflavins undergo facile photolysis. One major product was identified as 6-aminoflavin. A further product, which was formed also during acid decomposition of the azide, results from opening of the flavin benzene ring and is proposed to have a lumazine structure. 6-Thiocyanatoflavins are easily converted by dithiothreitol to 6-mercaptoflavins. The latter are stabilized against dimerization in the presence of reducing thiols. 6-Mercaptoflavins have a pK of 5.9, which corresponds to ionization of the 6-SH function. The neutral form is yellow, while the anion is green, due to a long-wavelength band (lambda max approximately 600 nm) extending beyond 700 nm. These properties suggest the use of these 6-substituted flavins for probing the active site of flavin enzymes. Because their reactive substituents are in close proximity to the flavin N(5)-position, these 6-substituted derivatives should also serve as useful probes of the environment around the flavin N(5), a position known to be involved in all flavin-mediated redox processes.
Binding Sites↗
The equilibration of reducing equivalents within milk xanthine oxidase.
The rate at which reducing equivalents equilibrate among the several oxidation-reduction active sites in xanthine oxidase has been investigated using a pH-jump technique in which partially reduced enzyme in dilute buffer is mixed with concentrated anaerobic buffer at a different pH in a conventional stopped flow apparatus. It is found that the rate constant associated with the observed spectral change varies with pH, doubling from 155 s-1 at pH 6 to 330 s-1 at pH 8.5, but is always found to be approximately 10-fold greater than kcat at the same pH. The observation of fast rates for the equilibration of reducing equivalents within xanthine oxidase is consistent with a great deal of indirect evidence from conventional kinetic studies of both the oxidative and reductive half-reactions of xanthine oxidase and lends support to the rapid equilibrium model that has been proposed for the oxidation-reduction interactions of the several centers in xanthine oxidase (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). The present conclusions are in conflict, however, with the interpretation of recent flash photolysis experiments with xanthine oxidase (Battacharyya, A., Tollin, G., Davis, M. D., and Edmondson, D. E. (1983) Biochemistry 22, 5270-5279). Possible sources for the apparent inconsistencies between the flash photolysis results and those of the present experiments are discussed.
Animals↗
Reactivity of old yellow enzyme with alpha-NADPH and other pyridine nucleotide derivatives.
The reaction of Old Yellow Enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. alpha-NADPH, beta-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 microM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both alpha- and beta-NADPH are capable of reducing OYE, and alpha-NADPH is more effective. Using beta-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with alpha-NADPH was significantly higher than that with beta-NADPH.
Chromatography, High Pressure Liquid↗
Interflavin oxidation-reduction reactions between pig kidney general acyl-CoA dehydrogenase and electron-transferring flavoprotein.
The mechanism of interflavin electron transfer between pig kidney general acyl-CoA dehydrogenase (GAD) and its physiological acceptor, electron-transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance and fluorescence measurements. At 3 degrees C, pH 7.6, reoxidation of the dehydrogenase (stoichiometrically reduced by octanoyl-CoA) by ETF is multiphasic, consisting of two rapid phases (t1/2 of about 20 and 50 ms), a slower phase half-complete in about 1 s, and a final reaction with a half-time of 20 s. Only the two most rapid phases are significant in turnover. This complicated reaction course was dissected by examining the rates of plausible individual steps, e.g., GAD2e X P + ETF1e, GAD1e X P + ETFox, and GAD1e X P + ETF1e (where P represents the product, octenoyl-CoA, and the subscripts indicate the redox state of the flavin). Rapid reaction and static fluorescence measurements, in all cases, showed that the final equilibrium mixture included appreciable levels of oxidized ETF. This was confirmed by measuring the reverse reactions, e.g., ETF1e + GADox X P, ETF1e + GAD1e X P, and ETF2e + GADox X P. These data support the following overall scheme for the reaction of GAD2e X P with ETFox: The first and second phases correspond to reoxidation of GAD2e X P in two successive one-electron steps requiring two molecules of ETFox. This results in a rapid rise in absorbance at 370 nm where the red anionic radicals of both product-complexed dehydrogenase and ETF absorb strongly.(ABSTRACT TRUNCATED AT 250 WORDS)
Acyl-CoA Dehydrogenases↗
Potentiometric studies of native and flavin-substituted Old Yellow Enzyme.
We have measured the redox potentials for the flavin cofactor of native Old Yellow Enzyme and for a series of chemically modified flavin derivatives bound to the apoprotein. These flavin derivatives have midpoint potentials ranging from -120 to -300 mV in free solution. For the native enzyme, the midpoint potential of the first one-electron couple EFMNox + e- in equilibrium EFMN-. is E1 = -245 +/- 5 mV and that for the second one-electron couple EFMN-. + e- in equilibrium EFMN red is E2 = -215 +/- 5 mV in 0.1 M phosphate buffer at pH 7.0 and 25 degrees C. Thus, the apoprotein lowers the two-electron midpoint potential of FMN below its value in free solution (Emid,bound = -230 mV, Emid,free = -210 mV). A similar effect on the two-electron midpoint potentials of most of the chemically modified flavins is observed upon binding to the apoprotein. Therefore, the relative order of the respective midpoint potentials of this series of flavins is maintained upon binding to the apoenzyme. However, the effect of the apoprotein on the separation between E1 and E2 varies considerably over this series of flavin derivatives, resulting in quite different levels of thermodynamic stability for the one-electron-reduced (semiquinone) forms of the flavin-substituted enzymes. The optical absorption spectra of these flavin-substituted enzymes were determined in the presence of several phenolic compounds which are known to bind to the native enzyme and to give characteristic long wavelength transitions. We have found that the positions of the wavelength maxima of these transitions are shifted to longer wavelengths as the measured redox potentials of the enzyme-bound flavins are increased. The relationship between the energy of these long wavelength transitions (expressed as vCT = 1/lambda max) and the redox potential of the enzyme-bound flavin is interpreted as further evidence that these transitions arise from charge-transfer complexes between the phenolic compounds and the flavin.
Dithionite↗
The reaction of arsenite-complexed xanthine oxidase with oxygen. Evidence for an oxygen-reactive molybdenum center.
The effects of arsenite on the reaction of reduced xanthine oxidase with oxygen are determined. The kinetics of the reaction monitoring the return of enzyme absorbance are investigated as are the kinetics and stoichiometries of peroxide and superoxide formation. Although some of the effects of arsenite are qualitatively consistent with expectations based on the known perturbation of the molybdenum midpoint potentials by arsenite, several results cannot be so easily explained. Specifically, arsenite introduces a very rapid phase (kobs = 110 s-1 at 125 microM oxygen) to the oxidative half-reaction which is not observed with the native enzyme. Arsenite also diminishes the amount of superoxide produced and eliminates one-electron reduced enzyme as a detectable kinetic intermediate in the reoxidation pathway. These differences appear to result from the ability of arsenite to greatly enhance the oxygen- and/or superoxide-reactivity of the reduced molybdenum center. This is reflected in the observation that reduced forms of arsenite-complexed xanthine oxidase lacking functional FAD (iodoacetamide-alkylated enzyme and deflavo enzyme) react relatively rapidly with oxygen whereas these reactions are quite slow in the absence of arsenite.
Arsenic↗
8-Azidoflavins as photoaffinity labels for flavoproteins.
8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.
Affinity Labels↗
Effect of substrate and pH on the oxidative half-reaction of phenol hydroxylase.
The oxidative half-reaction of phenol hydroxylase has been studied by stopped-flow spectrophotometry. Three flavin-oxygen intermediates can be detected when the substrate is thiophenol, or m-NH2, m-OH, m-CH3, m-Cl, or p-OH phenol. Intermediate I, the flavin C(4a)-hydroperoxide, has an absorbance maximum at 380-390 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate III, the flavin C(4a)-hydroxide, has an absorbance maximum at 365-375 nm and an extinction coefficient approximately 10,000 M-1 cm-1. Intermediate II has absorbance maxima of 350-390 nm and extinction coefficients of 10,000-16,000 M-1 cm-1 depending on the substrate. A Hammett plot of the logarithm of the rates of the oxygen transfer step, the conversion of intermediate I to intermediate II, gives a straight line with a slope -0.5. Fluoride ion is a product of the enzymatic reaction when 2,3,5,6-tetrafluorophenol is the substrate. These results are consistent with an electrophilic substitution mechanism for oxygen transfer. The conversions of I to II and II to III are acid-catalyzed. A kinetic isotope effect of 8 was measured for the conversion of II to III using deuterated resorcinol as substrate. The conversion of III to oxidized enzyme is base-catalyzed, suggesting that the reaction depends on the removal of the flavin N(5) proton. Product release occurs at the same time as the formation of intermediate III, or rapidly thereafter. The results are interpreted according to the ring-opened model of Entsch et al. (Entsch, B., Ballou, D. P., and Massey, V. (1976) J. Biol. Chem. 251, 2550-2563).
Chemical Phenomena↗
Characterization of arsenite-complexed xanthine oxidase at room temperature. Spectral properties and pH-dependent redox behavior of the molybdenum-arsenite center.
Several aspects of the interaction of xanthine oxidase with arsenite are investigated. Room temperature potentiometric titrations using EPR to monitor Molybdenum reduction reveal midpoint potentials of -225 mV for the Mo(VI)-arsenite/Mo(V)-arsenite couple and -440 mV for the Mo(V)-arsenite/Mo(IV)-arsenite couple at pH 8.3. Under the same conditions, the values for native enzyme are -395 mV and -420 mV, respectively. The predicted effects of the altered Mo(VI)/Mo(V) potential on the distributions of reducing equivalents in partially reduced enzyme are compared with the experimentally observed effects in optical experiments. The bleaching that occurs on reduction of the chromophore that is generated when arsenite binds to oxidized enzyme is characterized and found to be associated with reduction of Mo(V)-arsenite to Mo(V)-arsenite. This probe enables determination of the midpoint potential for this conversion using optical data. From such data at a series of pH values ranging from 6.15 to 9.9, a pH dependence of -60 mV/pH unit increase is determined for this couple above pH 7. The ability of arsenite to bind to reduced xanthine oxidase and to desulfo enzyme are also investigated. Reduced active enzyme binds arsenite much more tightly (Kd less than 0.1 microM) and more rapidly than does oxidized active enzyme (Kd = 8 microM); oxidized desulfo enzyme binds arsenite almost as tightly (Kd = 20 microM) as does the oxidized active enzyme.
Animals↗
4-Thioflavins as active site probes of flavoproteins. Reactions with sulfite.
4-Thioflavins react with sulfite under aerobic conditions to yield highly fluorescent products with absorption maxima around 410 nm. These products have been identified as 4-hydroxy-4-sulfonylflavins, and have been shown to arise from a series of reactions following the O2-dependent reoxidation of an intermediate with absorption maxima at 363 and 465 nm. Under anaerobic conditions, the same intermediate is formed, but decays to a 350 nm absorbing species, which is probably the N(5)-sulfite adduct of 4-thioflavin. A plausible mechanism is described for the formation of the derivatives, and several of their chemical and physical properties are described. Distinctly different results between different proteins are obtained when sulfite reacts with enzyme-bound 4-thioflavins. 4-Thio-FAD-D-amino acid oxidase and 4-thio-FMN-lactate oxidase react rapidly to yield the N(5)-sulfite adducts, as occurs with the native enzymes. 4-Thio-FAD-p-hydroxybenzoate hydroxylase reacts slowly in a manner paralleling the reaction with the free 4-thioflavins.
4-Hydroxybenzoate-3-Monooxygenase↗
pH dependence of the reoxidation of p-hydroxybenzoate hydroxylase 2,4-dihydroxybenzoate complex.
Oxidation of reduced p-OH-benzoate hydroxylase . 2,4-diOH-benzoate complex by molecular oxygen occurs in four clearly defined steps. Inclusion of azide in the reaction medium slows the rate for each step. We have found that the rates for the last three steps as well as the UV/visible absorbance spectrum for the second intermediate are markedly dependent on pH. Formation of intermediate II is base-catalyzed while its decay to intermediate III is acid-catalyzed. Formation of oxidized enzyme from intermediate III is base-catalyzed. The spectrum of intermediate II is shifted to longer wavelengths on raising the pH. A pKa of about 7.8 can be calculated for each of these pH-dependent transitions, except for the rate of formation of oxidized enzyme. This rate appears to be directly dependent on hydroxide ion concentration. The presence of azide does not change the observed pH dependence. These results are discussed in the context of a 4a,5-ring opened flavin structure for intermediate II.
4-Hydroxybenzoate-3-Monooxygenase↗
Effect of monovalent anions on the mechanism of phenol hydroxylase.
The mechanism of phenol hydroxylase (EC 1.14.13.7) has been studied by steady state and rapid reaction kinetic techniques. Both techniques give results consistent with the Bi Uni Uni Bi ping-pong mechanism proposed for other flavin-containing aromatic hydroxylases. The enzyme binds phenolic substrate and NADPH in that order, followed by reduction of the flavin and release of NADP+. A transient charge transfer complex between reduced enzyme and NADP+ can be detected. Molecular oxygen then reacts with the reduced enzyme-substrate complex. Two to three flavin-oxygen intermediates can be detected in the oxidative half-reaction depending on the substrate, provided monovalent anions are present. Oxygen transfer is complete with the formation of the second intermediate. Based on its UV absorption spectrum and on the fact that oxygen transfer has taken place, the last of these intermediates is presumably the flavin C(4a)-hydroxide. Monovalent anions are uncompetitive inhibitors of phenol hydroxylase. The mechanistic step most affected is the dehydration of the flavin C(4a)-hydroxide to give oxidized enzyme. Chloride also kinetically stabilizes the blue flavin semiquinone of phenol hydroxylase during photoreduction. These data suggest binding of monovalent anions results in stabilization of a proton on the N(5) position of the flavin.
Anions↗
4-Thioflavins as active site probes of flavoproteins. General properties.
4-Thioflavins (oxygen at position 4 replaced by sulfur) have been studied as potential active site probes of flavoproteins. They react readily with thiol reagents, with large spectral changes, which should be useful for testing the accessibility of the flavin 4-position in flavoproteins. They have an oxidation-reduction potential at pH 7 of -0.055 V, approximately 0.15 V higher than that of native flavins. The spectral characteristics in the fully reduced state show two clear absorption bands, dependent on the ionization state (pK = 4.5). The lowest energy band of the neutral dihydroflavin has a maximum at approximately 485 nm while that of the anion is approximately 425 nm. This should be useful in defining the ionization state of the reduced flavin in flavoproteins. The spectral characteristics of the semiquinoid forms of 4-thioflavins have been determined bound to the apoproteins of flavodoxin and D-amino acid oxidase. The neutral radical has an absorption maximum at 730 nm, while the anion radical has an unusually sharp peak at 415 nm. The reduced forms of 4-thioflavins, free and enzyme bound, react with O2 to regenerate oxidized 4-thioflavin. Reduced 4-thio-FAD p-hydroxybenzoate hydroxylase, however, in its reaction with O2, undergoes a substantial conversion to the native FAD-enzyme. 4-Thioflavins are unusually susceptible to attack by nucleophiles such as hydroxylamine and amines to form the respective 4-hydroxyimino- and 4-aminoflavins, offering the possibility of forming stable covalent flavin-protein linkages with suitably positioned protein residues. Thiols also react with 4-thioflavins, promoting their conversion to the normal (4-oxo) flavin coenzymes. Such reactivity has been found with the apoenzymes of glucose oxidase and lactate oxidase, providing evidence for a thiol residue in the active site of these enzymes.
4-Hydroxybenzoate-3-Monooxygenase↗
Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride.
Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)
Acyl Coenzyme A↗
Methylation of the active center histidine 217 in D-amino acid oxidase by methyl-p-nitrobenzenesulfonate.
Incubation of D-amino acid oxidase with excess methyl-p-nitrobenzenesulfonate results in a pseudo-first order, irreversible loss of 95% of the assayable activity using D-phenylglycine as substrate. The rate of inactivation reaches a limiting value of 0.021 min-1 (pH 7.7, 22 degrees C) as the concentration of inhibitor is increased, consistent with a two-step mechanism with complex formation prior to covalent inactivation. This rate decreases with decreasing pH in a manner that suggests that the basic form of a group within the enzyme, with an apparent pKa of 6.7, is required for inactivation. Approximately three [14C]methyl residues are covalently incorporated at maximal inactivation. The competitive inhibitor benzoate prevents inactivation and the incorporation of approximately 1 methyl residue. Histidine 217, the residue modified by 5-dimethylaminonaphthalene-1-sulfonyl chloride (Swenson, R. P., Williams, C. H., Jr., and Massey, V. (1983) J. Biol. Chem. 258, 497-502), was identified as the major residue modified in the absence of benzoate. Both the 1-methyl and 3-methyl derivatives of histidine 217 were observed in a mole ratio of 0.64:0.36.
Amino Acids↗