Structural and kinetic studies on the Ser101Ala variant of choline oxidase: Catalysis by compromise

The oxidation of choline catalyzed by choline oxidase includes two reductive half-reactions where FAD is reduced by the alcohol substrate and by an aldehyde intermediate transiently formed in the reaction. Each reductive half-reaction is followed by an oxidative half-reaction where the reduced flavin is oxidized by oxygen. Here, we have used mutagenesis to prepare the Ser101Ala mutant of choline oxidase and have investigated the impact of this mutation on the structural and kinetic properties of the enzyme. The crystallographic structure of the Ser101Ala enzyme indicates that the only differences between the mutant and wild-type enzymes are the lack of a hydroxyl group on residue 101 and a more planar configuration of the flavin in the mutant enzyme. Kinetics established that replacement of Ser101 with alanine yields a mutant enzyme with increased efficiencies in the oxidative half-reactions and decreased efficiencies in the reductive half-reactions. This is accompanied by a significant decrease in the overall rate of turnover with choline. Thus, this mutation has revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase, which requires fine-tuning of four consecutive half-reactions for the conversion of an alcohol to a carboxylic acid.     

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.     

Electron transfer in milk xanthine oxidase as studied by pulse radiolysis

Electron transfer within milk xanthine oxidase has been examined by the technique of pulse radiolysis. Radiolytically generated N-methylnicotinamide radical or 5-deazalumiflavin radical has been used to rapidly and selectively introduce reducing equivalents into the enzyme so that subsequent equilibration among the four redox-active centers of the enzyme (a molybdenum center, two iron-sulfur centers, and FAD) could be monitored spectrophotometrically. Experiments have been performed at pH 6 and 8.5, and a comprehensive scheme describing electron equilibration within the enzyme at both pH values has been developed. All rate constants ascribed to equilibration between specific pairs of centers in the enzyme are found to be rapid relative to enzyme turnover under the same conditions. Electron equilibration between the molybdenum center and one of the iron-sulfur centers of the enzyme (tentatively assigned Fe/S I) is particularly rapid, with a pH-independent first-order rate constant of approximately 8.5 x 10(3) s-1. The results unambiguously demonstrate the role of the iron-sulfur centers of xanthine oxidase in mediating electron transfer between the molybdenum and flavin centers of the enzyme.     

Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.     

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.     

Mechanistic aspects of the covalent flavoprotein dimethylglycine oxidase of Arthrobacter globiformis studied by stopped-flow spectrophotometry

Dimethylglycine oxidase (DMGO) is a covalent flavoenzyme from Arthrobacter globiformis that catalyzes the oxidative demethylation of dimethylglycine to yield sarcosine, formaldehyde, and hydrogen peroxide. Stopped-flow and steady-state kinetic studies have been used to study the reductive and oxidative half-reactions using dimethylglycine and O2 as substrates. The reductive half-reaction is triphasic. The rate of the fast phase is dependent on substrate concentration, involves flavin reduction, and has a limiting rate constant of 244 s(-1). This phase also displays a kinetic isotope effect of 2.9. Completion of the first kinetic phase generates an intermediate with broad spectral signature between 350 and 500 nm, which is attributed to a reduced enzyme-iminium charge-transfer species, similar to the purple intermediate that accumulates in reactions of D-amino acid oxidase (DAAO) with alanine. The second phase (16 s(-1)) is independent of substrate concentration and is attributed to iminium hydrolysis/deprotonation. The third phase (2 s(-1)) is attributed to product release, the rate of which is less than the steady-state turnover rate (10.6 s(-1)). Flavin oxidation of dithionite- and dimethylglycine-reduced enzyme by O2 occurs in a single phase, and the rate shows a linear dependence on oxygen concentration, giving bimolecular rate constants of 342 and 201 mM(-1) x s(-1), respectively. Enzyme-monitored turnover experiments indicate that decay of the reduced enzyme-iminium intermediate is rate-limiting, consistent with rate constants determined from single turnover studies. A minimal kinetic mechanism is presented, which establishes a close relationship to the mechanism of action of DAAO. The covalent flavin in dimethylglycine oxidase is identified as an alphaN1-histidyl48-FAD, and equilibrium titration studies establish a single redox center that displays typical flavoprotein 'oxidase' characteristics.     

The radical chemistry of milk xanthine oxidase as studied by radiation chemistry techniques

The kinetics of electron transfer within the molybdoflavoenzyme xanthine oxidase has been investigated using the technique of pulse radiolysis. Subsequent to one-electron reduction of native enzyme at 20 degrees C in 20 mM pyrophosphate buffer, pH 8.5, using the CO-.2 species as reductant, a spectral change is observed having a rate constant of approximately 290 s-1. From its wavelength dependence, this spectral change is assigned to the transfer of an electron from flavin semiquinone (formed on reaction with the CO2-. species) to one of the iron-sulfur centers of the enzyme in an intramolecular equilibration process. The value for this rate constant agrees well with the 330 s-1 observed in previous stopped-flow pH-jump experiments carried out at 25 degrees C (Hille, R., and Massey, V. (1986) J. Biol. Chem. 261, 1241-1247). Experimental results with fully reduced enzyme reacting with the radiolytically generated N.3 species also support the conclusion that the equilibration of reducing equivalents among the oxidation-reduction centers of xanthine oxidase is a rapid process. Evidence is also found that xanthine oxidase possesses an unusually reactive disulfide bond that is reduced rapidly by radiolytically generated radicals. The ramifications of the present results with regard to the interpretation of experiments involving chemically reactive radical species, generated either by photolysis or radiolysis, are discussed.     

Erv2p: characterization of the redox behavior of a yeast sulfhydryl oxidase

The FAD prosthetic group of the ERV/ALR family of sulfhydryl oxidases is housed at the mouth of a 4-helix bundle and communicates with a pair of juxtaposed cysteine residues that form the proximal redox active disulfide. Most of these enzymes have one or more additional distal disulfide redox centers that facilitate the transfer of reducing equivalents from the dithiol substrates of these oxidases to the isoalloxazine ring where the reaction with molecular oxygen occurs. The present study examines yeast Erv2p and compares the redox behavior of this ER luminal protein with the augmenter of liver regeneration, a sulfhydryl oxidase of the mitochondrial intermembrane space, and a larger protein containing the ERV/ALR domain, quiescin-sulfhydryl oxidase (QSOX). Dithionite and photochemical reductions of Erv2p show full reduction of the flavin cofactor after the addition of 4 electrons with a midpoint potential of -200 mV at pH 7.5. A charge-transfer complex between a proximal thiolate and the oxidized flavin is not observed in Erv2p consistent with a distribution of reducing equivalents over the flavin and distal disulfide redox centers. Upon coordination with Zn2+, full reduction of Erv2p requires 6 electrons. Zn2+ also strongly inhibits Erv2p when assayed using tris(2-carboxyethyl)phosphine (TCEP) as the reducing substrate of the oxidase. In contrast to QSOX, Erv2p shows a comparatively low turnover with a range of small thiol substrates, with reduced Escherichia coli thioredoxin and with unfolded proteins. Rapid reaction studies confirm that reduction of the flavin center of Erv2p is rate-limiting during turnover with molecular oxygen. This comparison of the redox properties between members of the ERV/ALR family of sulfhydryl oxidases provides insights into their likely roles in oxidative protein folding.     

Lys300 plays a major role in the catalytic mechanism of maize polyamine oxidase

Maize polyamine oxidase (MPAO) is a flavin adenine dinucleotide (FAD)-dependent enzyme that catalyses the oxidation of spermine and spermidine at the secondary amino groups. The structure of MPAO indicates a 30-A long U-shaped tunnel that forms the catalytic site, with residues Glu62 and Glu170 located close to the enzyme-bound FAD and residue Tyr298 in close proximity to Lys300, which in turn is hydrogen-bonded to the flavin N(5) atom via a water molecule (HOH309). To provide insight into the role of these residues in the catalytic mechanism of FAD reduction, we have performed steady-state and stopped-flow studies with wild-type, Glu62Gln, Glu170Gln, Tyr298Phe, and Lys300Met MPAO enzymes. We show that the steady-state enzyme activity is governed by an ionisable group with a macroscopic pK(a) of approximately 5.8. Kinetic analysis of the Glu62Gln, Glu170Gln, and Tyr298Phe MPAO enzymes have indicated (i) only small perturbations in catalytic activity as a result of mutation and (ii) steady-state pH profiles essentially unaltered when compared to the wild-type enzyme, suggesting that these residues do not play a critical role in the reaction mechanism. These kinetic observations are consistent with computational calculations that suggest that Glu62 and Glu170 are protonated over the pH range accessible to kinetic studies. Substitution of Lys300 with Met in MPAO resulted in a 1400-fold decrease in the rate of flavin reduction and a 160-fold decrease in the equilibrium dissociation constant for the Lys300Met-spermidine complex, consistent with a major role for this residue in the mechanism of substrate oxidation. A sizable solvent isotope effect (SIE = 5) accompanies FAD reduction in the wild-type enzyme and steady-state turnover (SIE = 2.3) of MPAO, consistent with the reductive half-reaction of MPAO making a major contribution to rate limitation in steady-state turnover. Studies using the enzyme-monitored turnover method indicate that oxidized FAD is the prominent form during steady-state turnover, consistent with the reductive half-reaction being rate-limiting. Our studies indicate the importance of Lys300 and probable importance of HOH309 to the mechanism of flavin reduction in MPAO. Possible roles for Lys300 and water in the mechanism of flavin reduction are discussed.     

Dihydroorotate dehydrogenase B of Enterococcus faecalis. Characterization and insights into chemical mechanism.

Enterococcus faecalis dihydroorotate dehydrogenase B is a heterodimer of 28 and 33 kDa encoded by the pyrK and pyrDb genes. Both subunits copurify during all chromatographic steps, and, as determined by HPLC, one FMN and one FAD are bound per heterodimer. The enzyme catalyzes efficient oxidation of 4-S-NADH by orotate. Isotope effect and pH data suggest that reduction of flavin by NADH at the PyrK site is only partially rate limiting with no kinetically significant proton transfer occurring in the reductive half-reaction; therefore, a group exhibiting a pK of 5.7 +/- 0.2 represents a residue involved in binding of NADH rather than in catalysis. The reducing equivalents are shuttled between the NADH-oxidizing flavin in PyrK and the orotate-reacting flavin in PyrDb, by iron-sulfur centers through flavin semiquinones as intermediates. A solvent kinetic isotope effect of 2.5 +/- 0.2 on V is indicative of rate-limiting protonation in the oxidative half-reaction and most likely reflects the interaction between the isoalloxazine N1 of the orotate-reducing flavin and Lys 168 (by analogy with L. lactis DHODase A). The oxidative half-reaction is facilitated by deprotonation of the group(s) with pK(s) of 5.8-6.3 and reflects either deprotonation of the reduced flavin or binding of orotate; this step is followed by hydride transfer to C6 and general acid-assisted protonation (pK of 9.1 +/- 0.2) at C5 of the product.     

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.     

Egg white sulfhydryl oxidase: kinetic mechanism of the catalysis of disulfide bond formation

The flavin-dependent sulfhydryl oxidase from chicken egg white catalyzes the oxidation of sulfhydryl groups to disulfides with reduction of oxygen to hydrogen peroxide. The oxidase contains FAD and a redox-active cystine bridge and accepts a total of 4 electrons per active site. Dithiothreitol (DTT; the best low molecular weight substrate known) reduces the enzyme disulfide bridge with a limiting rate of 502/s at 4 degrees C, pH 7.5, yielding a thiolate-to-flavin charge-transfer complex. Further reduction to EH4 is limited by the slow internal transfer of reducing equivalents from enzyme dithiol to oxidized flavin (3.3/s). In the oxidative half of catalysis, oxygen rapidly converts EH4 to EH2, but Eox appearance is limited by the slow internal redox equilibration. During overall turnover with DTT, the thiolate-to-flavin charge-transfer complex accumulates with an apparent extinction coefficient of 4.9 mM-1 cm-1 at 560 nm. In contrast, glutathione (GSH) is a much slower reductant of the oxidase to the EH2 level and shows a kcat/Km 100-fold smaller than DTT. Full reduction of EH2 by GSH shows a limiting rate of 3.6/s at 4 degrees C comparable to that seen with DTT. Reduced RNase is an excellent substrate of the enzyme, with kcat/Km per thiol some 1000- and 10-fold better than GSH and DTT, respectively. Enzyme-monitored steady-state turnover shows that RNase is a facile reductant of the oxidase to the EH2 state. This work demonstrates the basic similarity in the mechanism of turnover between all of these three substrates. A physiological role for sulfhydryl oxidase in the formation of disulfide bonds in secreted proteins is discussed.     

The presence of a reducible disulfide bond in milk xanthine oxidase

The stoichiometry of reducing equivalents per protomer for the complex molybdoflavoprotein xanthine oxidase has been re-examined by reductive titrations with sodium dithionite and anaerobic reoxidation with cytochrome c and phenazine methosulfate of dithionite- or photo-reduced enzyme. It is found that 8.0 +/- 0.1 reducing equivalents are taken up (or given up) by the enzyme, a value of 2 eq greater than expected on the basis of the known oxidation-reduction centers in the enzyme. The reaction of reduced xanthine oxidase with [14C]iodoacetate indicates that, in the reduced form of the enzyme, additional cysteine residues are available for reaction. These results, in conjunction with the observation that reaction of oxidized enzyme with sulfite results in the appearance of an additional equivalent of thiol capable of reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) or iodoacetate, indicate the presence of a disulfide linkage in the enzyme that can be reduced by dithionite or photochemically employing EDTA and 5-deazaflavin. Neither xanthine nor lumazine, however, is capable of reducing this oxidation-reduction center, suggesting that the disulfide does not play a role in the catalytic reactions of the enzyme. These results resolve discrepancies in the literature which indicated that greater than 6 reducing equivalents were consistently needed to bring about the complete reduction of xanthine oxidase.     

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.     

Purification and characterization of multiple forms of rat liver xanthine oxidoreductase expressed in baculovirus-insect cell system

cDNA of rat liver xanthine oxidoreductase (XOR), a molybdenum-containing iron-sulfur flavoprotein, was expressed in a baculovirus-insect cell system. The expressed XOR consisted of a heterogeneous mixture of native dimeric, demolybdo-dimeric, and monomeric forms, each of which was separated and purified to homogeneity. All the expressed forms contained flavin, of which the semiquinone form was stable during dithionite titration after dithiothreitol treatment, indicating that the flavin domains of all the expressed molecules have the intact conformations interconvertible between NAD(+)-dependent dehydrogenase (XDH) and O(2)-dependent oxidase (XO) types. The absorption spectrum and metal analyses showed that the monomeric form lacks not only molybdopterin but also one of the iron-sulfur centers. The reductive titration of the monomer with dithionite showed that the monomeric form required only three electrons for complete reduction, and the redox potential of the iron-sulfur center in the monomeric form is a lower value than that of FAD. In contrast to native or demolybdo-dimeric XDHs, the monomer showed a very slow reductive process with NADH under anaerobic conditions, although the conformation around FAD is a dehydrogenase form, suggesting the important role of the iron-sulfur center in the reductive process of FAD with the reduced pyridine nucleotide.     

Flavin analogs as mechanistic probes of adrenodoxin reductase-dependent electron transfer to the cholesterol side chain cleavage cytochrome P-450 of the adrenal cortex

The intrinsic isotope effect on the reduction of the FAD-containing dehydrogenase electron transferase, adrenodoxin reductase, by (4S)-[2H]NADPH has been determined to be 7.1 to 7.7. The replacement of FAD by a series of FAD analogs at the active site of adrenodoxin reductase with oxidation-reduction potentials which vary over a range of 212 mV has made it possible to extrapolate to this limiting value from the variation in the observed isotope effect on Vmax with flavin midpoint potential. Stop-flow studies which allow the direct determination of the intrinsic isotope effect on the reductive half-reaction corroborate this result. During the steady state reduction of ferricyanide by the native enzyme under conditions of Vmax, this isotope effect is almost fully expressed (VH/VD = 6.7 to 6.8). In contrast, we observe a dramatic attenuation of the intrinsic isotope effect (due to hydride transfer to flavin) when the oxidative half-reaction is mediated by the natural acceptor protein, the 2Fe/2S ferredoxin, adrenodoxin. In a coupled three-protein system, the adrenodoxin-mediated reductions of both the artificial electron acceptor, cytochrome c, and the physiological electron acceptor, cytochrome P-450scc, by adrenodoxin reductase occur at similar rates and with similar kinetic isotope effects (1.9 to 2.0) when (4S)-[2H]NADPH is the reductant. We infer similar mechanisms for the reduction of both cytochromes. These results are in agreement with previous studies (Lambeth, J.D., and Kamin, H. (1979) J. Biol. Chem. 254, 2766-2774) which show that the reductive half-reaction is not solely rate-determining in adrenodoxin-mediated processes. The observation of a linear free energy relationship between Vmax and the flavin midpoint potential during steady state reduction of ferricyanide confirms that the reductive half-reaction is rate-determining in this assay. The relationship between Vmax and flavin midpoint potential in reactions which require adrenodoxin suggests that the midpoint potential of native adrenodoxin reductase has been optimized. Thus, the apoenzyme of adrenodoxin reductase tailors the midpoint potential of bound FAD in order to balance the activation energies of the reductive and oxidative half-reactions.     

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.     

High-level expression of human liver monoamine oxidase A in Pichia pastoris: comparison with the enzyme expressed in Saccharomyces cerevisiae.

The high-level expression, purification, and characterization of recombinant membrane-bound human liver monoamine oxidase A (MAO-A) in Pichia pastoris is described. Two liters of fermentation culture produces 1170 units (660 mg) of MAO-A. The enzyme is purified in a 35% yield, is homogeneous on denaturing gel electrophoresis, and exhibits a single species (60,512 +/- 6 Da) on electrospray mass spectrometry. It contains 1 mol of 8alpha-S-cysteinyl FAD/mole of enzyme and exhibits >95% functionality. In contrast, the Saccharomyces cerevisiae-expressed enzyme is partially processed by C-terminal serine removal as demonstrated by mass spectra. The amino termini of both P. pastoris- and S. cerevisiae-expressed MAO-A are acetylated on the N-terminal methionine. The steady-state kinetic properties of P. pastoris-expressed MAO-A are similar to those of S. cerevisiae-expressed MAO-A using the following substrates: phenethylamine, p-CF(3)-benzylamine, dopamine, serotonin, and kynuramine. Reductive titrations demonstrate that the recombinant enzyme is reduced by 1 mol of substrate or dithionite as expected for the two electron equivalents required for flavin reduction. Absorption and EPR spectra show no radical species in the resting enzyme while the anionic flavin radical is formed in 50% yield during the reductive titration with dithionite. These data demonstrate significant advantages in the heterologous expression of human MAO-A in P. pastoris compared with the published S. cerevisiae system in higher expression level (329 mg/L) and in a higher level of homogeneity of the isolated enzyme.     

Altered balance of half-reactions in p-hydroxybenzoate hydroxylase caused by substituting the 2'-carbon of FAD with fluorine

Apo-p-hydroxybenzoate hydroxylase was reconstituted using 2'-fluoro-2'-deoxy-arabino-FAD, a synthetic flavin in which the hydroxyl of the 2'-center of the ribityl chain was replaced with fluorine in an inverted configuration. The absorbance spectral changes caused by the binding of either p-hydroxybenzoate (pOHB) or 2,4-dihydroxybenzoate (2,4-diOHB) indicated that the isoalloxazine of the artificial flavin adopts the more solvent-exposed "out" conformation rather than the partially buried "in" conformation near the aromatic substrate. In contrast, the flavin of the natural enzyme adopts the in conformation when pOHB is bound. Much of the behavior of the artificial enzyme can be rationalized in light of the preference of the flavin for the out conformation, including the weaker binding of pOHB, the tighter binding of 2,4-diOHB, and the slower reactions involved in the hydroxylation of pOHB and 2,4-diOHB. Particularly noteworthy is the enhancement of the reduction of the flavin by NADPH when pOHB is bound to the active site, consistent with the recent finding that the reaction occurs when the flavin adopts the out conformation (Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158). Thus, whereas the change that induces the out conformation is detrimental to the oxidative half-reaction, it improves the reductive half-reaction, showing that the control of the flavin position in p-hydroxybenzoate hydroxylase represents a compromise between the conflicting needs of two chemically disparate half-reactions, and demonstrating that the 2'-hydroxyl of FAD can serve as a critical control element in flavoenzyme catalysis.     

The redox centers of xanthine oxidase are on independent structural domains of the enzyme

Xanthine oxidase employs four electron transport sites (flavin adenine dinucleotide (FAD), molybdenum, and two FeS centers) in catalyzing a variety of redox reactions. To determine whether the redox sites reside in independent domains of the enzyme, the temperature of heat inactivation of each site's catalytic activity was determined, except that no attempt was made to distinguish between the two FeS sites. In the oxidase form of xanthine oxidase, the order of thermal stabilities was Mo greater than FAD greater than FeS, while after conversion to its dehydrogenase form the above ranking was Mo greater than FeS greater than FAD. The small but reproducible difference in heat inactivation temperatures among the redox sites demonstrated that the sites are located in separate domains of the enzyme. To confirm the above segregation of redox centers, the temperature of heat-induced release of each redox cofactor from its site on the enzyme was examined. These temperatures were found to be different for each redox cofactor and agreed closely with the heat inactivation temperatures measured above. The data thus demonstrate that both heat inactivation and cofactor release derive from thermal unfolding of independent domains. Using a technique termed "thermal digestion analysis," the FAD domain was located in a approximately equal to 42,000-Da tryptic fragment, while the FeS and Mo domains were isolated in a trypsin-resistant 92,000-Da fragment. We conclude that xanthine oxidase is constructed in modular fashion with the redox sites located in independent structural domains.