Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme. It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH. However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme. The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction. We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction.     

Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates p-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur. Original research papers (principally from the past 15 years) provide the information that is used to develop a comprehensive overview of the catalytic process. Much of this information has come from detailed analysis of many specific mutants of the enzyme using rapid reaction technology, biophysical measurements, and high-resolution structures obtained by X-ray crystallography. We describe how three conformations of the enzyme provide a foundation for the catalytic cycle. One conformation has a closed active site for the conduct of the oxygen reactions, which must occur in the absence of solvent. The second conformation has a partly open active site for exchange of substrate and product, and the third conformation has a closed protein structure with the isoalloxazine ring rotated out to the surface for reaction with NADPH, which binds in a surface cleft. A fundamental feature of the enzyme is a H-bond network that connects the phenolic group of the substrate in the buried active site to the surface of the protein. This network serves to protonate and deprotonate the substrate and product in the active site to promote catalysis and regulate the coordination of conformational states for efficient catalysis.     

A rate-limiting conformational change of the flavin in p-hydroxybenzoate hydroxylase is necessary for ligand exchange and catalysis: studies with 8-mercapto- and 8-hydroxy-flavins

The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).     

para-Hydroxybenzoate hydroxylase containing 6-hydroxy-FAD is an effective enzyme with modified reaction mechanisms

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) from Pseudomonas fluorescens, was replaced by 6-hydroxy-FAD (an extra hydroxyl group on the carbon at position 6 of the isoalloxazine ring of FAD). The catalytic cycle of this modified enzyme was analyzed and compared to the function of native (FAD) enzyme. Transient state kinetic analyses of the multiple changes in the chemical state of the flavin were the principal methods used to probe the mechanism. Four known substrates of the native enzyme were used to probe the reaction. With the natural substrate, p-hydroxybenzoate, the 6-hydroxy-FAD enzyme activity was 12-15% of native enzyme, due to a slower release of product from the enzyme, and less than one product molecule was formed per NADPH oxidized, due to an increased rate of nonproductive decomposition of the transient peroxyflavin essential to the catalytic pathway. More extensive changes in mechanism were observed with the substrates, 2,4-dihydroxybenzoate and p-aminobenzoate. The results suggest that, during catalysis, when the reduced state of FAD is ready for oxygen reaction, the substrate is located below and close to the C-4a/N-5 edge of the isoalloxazine ring. The nature of the high extinction, transient state of flavin, formed upon transfer of oxygen to substrate is discussed. It is not a flavin cation, and is unlikely to be an oxygen-substituted analogue of N-3/C-4 dihydroflavin.     

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.     

Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.     

Catalytic function of tyrosine residues in para-hydroxybenzoate hydroxylase as determined by the study of site-directed mutants

The role of protein residues in activating the substrate in the reaction catalyzed by the flavoprotein p-hydroxybenzoate hydroxylase was studied. X-ray crystallography (Schreuder, H. A., Prick, P.A.J., Wieringa, R.K., Vriend, G., Wilson, K.S., Hol, W.G. J., and Drenth, J. (1989) J. Mol. Biol. 208, 679-696) indicates that Tyr-201 and Tyr-385 form a hydrogen bond network with the 4-OH of p-hydroxybenzoate. Therefore, site directed mutants were constructed, converting each of these tyrosines into phenylalanines. Spectral (visible and fluorescence) properties, reduction potentials, and binding constants are very similar to those of wild type, indicating that there are no major structural changes in the mutants. In the absence of substrate, the mutants and wild type exhibit similar pH-dependent changes in the FAD spectrum. However, the enzyme-substrate complex of Tyr-201----Phe lacks an ionization observed in both wild type and Tyr-385----Phe, which preferentially bind the phenolate form of substrates. Tyr-201----Phe shows no preference, indicating that Tyr-201 is required to ionize the substrate. The mutants have less than 6% the activity of the wild type enzyme. The effects on catalysis were studied by stopped flow techniques. Reduction of FAD by NADPH is slower by 10-fold in Tyr-201----Phe and 100-fold in Tyr-385----Phe. When the reduced Tyr-201----Phe-p-hydroxybenzoate complex reacts with oxygen, a long-lived flavin-C(4a)-hydroperoxide is observed, which slowly eliminates H2O2 with very little hydroxylation. Thus, the role of Tyr-201 is to activate the substrate by stabilizing the phenolate. Tyr-385----Phe reacts with oxygen to form 25% oxidized enzyme, and 75% flavin hydroperoxide, which successfully hydroxylates the substrate. This mutant also hydroxylates the product (3, 4-dihydroxybenzoate) to form gallic acid.     

Increased positive electrostatic potential in p-hydroxybenzoate hydroxylase accelerates hydroxylation but slows turnover

Para-hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. These different reactions are coordinated through conformational rearrangements of the isoalloxazine ring within the protein structure. In this paper, we examine the effect of increased positive electrostatic potential in the active site upon the catalytic process with the enzyme mutation, Glu49Gln. This mutation removes a negative charge from a conserved buried charge pair. The properties of the Glu49Gln mutant enzyme are consistent with increased positive potential in the active site, but the mutant enzyme is difficult to study because it is unstable. There are two important changes in the catalytic function of the mutant enzyme as compared to the wild-type. First, the rate of hydroxylation of p-hydroxybenzoate by the transiently formed flavin hydroperoxide is an order of magnitude faster than in the wild-type. This result is consistent with one function proposed for the positive potential in the active site-to stabilize the negative C-4a-flavin alkoxide leaving group upon heterolytic fission of the peroxide bond. However, the mutant enzyme is a poorer catalyst than the wild-type enzyme because (unlike wild-type) the binding of p-hydroxybenzoate is a rate-limiting process. Our analysis shows that the mutant enzyme is slow to interconvert between conformations required to bind and release substrate. We conclude that the new open structure found in crystals of the Arg220Gln mutant enzyme [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613] is integral to the process of binding and release of substrate from oxidized enzyme during catalysis.     

Reaction of 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase with hydrogen peroxide. Formation of a covalent flavin-protein linkage

Hydrogen peroxide reacts with 2-thio-FAD-reconstituted p-hydroxybenzoate hydroxylase to yield a long wavelength intermediate (lambda max = 360, 620 nm) which can be isolated in stable form on removal of excess H2O2. The blue flavin derivative slowly decays in a second peroxide-dependent reaction to yield a new flavin product lacking long wavelength absorbance (lambda max = 408, 472 nm). This final peroxide-modified enzyme binds p-hydroxybenzoate with a 10-fold lower affinity than does the native enzyme; furthermore, substrate binding leads to the inhibition of enzyme reduction by NADPH. Trichloroacetic acid treatment of the final peroxide-modified enzyme results in the quantitative conversion of the bound flavin to free FAD. However, gel filtration of the modified enzyme in guanidine hydrochloride at neutral pH leads to the co-elution of protein and modified flavin. The nondenatured peroxide product reacts rapidly with hydroxylamine to yield 2-NHOH-substituted FAD. These observations indicate that the secondary reaction of peroxide with the blue intermediate from 2-thio-FAD p-hydroxybenzoate hydroxylase results in the formation of an acid-labile covalent flavin-protein linkage within the enzyme active site, involving the flavin C-2 position.     

Use of free energy relationships to probe the individual steps of hydroxylation of p-hydroxybenzoate hydroxylase: studies with a series of 8-substituted flavins.

We report Hammett correlations, using 8-substituted flavins, to clarify the mechanism of hydroxylation by p-hydroxybenzoate hydroxylase (PHBH). The 8-position of the FAD isoalloxazine ring was chosen for modifications, because in PHBH it has minimal interactions with the protein, and it is accessible to solvent and away from the site of hydroxylation. Although two intermediates, a flavin-C4a-hydroperoxide and a flavin-C4a-hydroxide, are known to participate in hydroxylation, the mechanism of oxygen transfer remains controversial. Mechanisms as diverse as electrophilic aromatic substitution, diradical formation, and isoalloxazine ring opening have been proposed. In the studies reported here, it was possible to monitor spectrally each of the individual steps involved in hydroxylation, because the FAD cofactor acts as a reporter group. Thus, with PHBH, substituted separately with nine derivatives of FAD altered in the 8-position, quantitative structure-reactivity relationships (QSAR) have been applied to probe the mechanisms of formation of the flavin-C4a-hydroperoxide, the conversion to the flavin-C4a-hydroxide with concomitant oxygen transfer to the substrate, and the dehydration of the flavin-C4a-hydroxide to form oxidized FAD. The individual chemical steps in the mechanism of PHBH were not altered when using any of the modified flavins, and normal products were obtained; however, the rates of individual steps were affected, and depended on the electronic properties of the 8-substituent. Increased hydroxylation rates were observed when a more electrophilic flavin-C4a-hydroperoxide (i.e., with an electron-withdrawing substituent at the 8-position) is bound to PHBH. On the basis of QSAR analysis, we conclude that the mechanism of the hydroxylation step is best described by electrophilic aromatic substitution.     

Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin

The oxygen transfer to p-hydroxybenzoate catalyzed by p-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 8124-8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997) Biochemistry 36, 7548-7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender approximately 35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives approximately 1.8-fold increases in hydroxylation rates, compared with a > or =4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.     

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.     

Role of protein flexibility in the catalytic cycle of p-hydroxybenzoate hydroxylase elucidated by the Pro293Ser mutant

Proline 293 of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa is in a highly conserved region of the flavoprotein aromatic hydroxylases. It is thought to impart rigidity to the backbone, as it partially cradles the FAD in these hydroxylases. Thus, this residue has been substituted with serine by site-directed mutagenesis to investigate the importance of flexibility of the peptide segment in catalysis. Differential scanning calorimetry demonstrated that the mutation has decreased the stability of the folded mutant protein compared to the wild-type PHBH. The increased flexibility in the protein backbone enhanced the accessibility of the flavin hydroperoxide intermediate to the solvent, causing an increase in the elimination of H(2)O(2) from this labile intermediate and, consequently, a decrease in the efficiency of substrate hydroxylation. Additionally, the increased accessibility of this mutant form of the enzyme makes it more susceptible than the wild-type enzyme to being trapped in the hydroxyflavin intermediate form in the presence of high levels of p-hydroxybenzoate. The mutation also lowers the pK(a) of the phenolic oxygen of bound p-hydroxybenzoate, and eliminates the pH dependence of the rate constant for flavin reduction by NADPH. These experimental observations lead to a model that explains how the wild-type protein can sense the charge of the 4-substituent of the aromatic ligand and link this charge to a flavin conformational change that is required for reaction with NADPH: (i) The peptide oxygen of Pro 293 is repelled by the negative charge of the phenolic oxygen of p-hydroxybenzoate. (ii) This repulsion is transmitted through the peptide backbone, causing the movement of Asn 300. (iii) The change in the position of Asn 300 triggers the movement of the flavin from the largely buried "in" conformation to the exposed, reactive "out" conformation.     

Analysis of the active site of the flavoprotein p-hydroxybenzoate hydroxylase and some ideas with respect to its reaction mechanism

The flavoprotein p-hydroxybenzoate hydroxylase has been studied extensively by biochemical techniques by others and in our laboratory by X-ray crystallography. As a result of the latter investigations, well-refined crystal structures are known of the enzyme complexed (i) with its substrate p-hydroxybenzoate and (ii) with its reaction product 3,4-dihydroxybenzoate and (iii) the enzyme with reduced FAD. Knowledge of these structures and the availability of the three-dimensional structure of a model compound for the reactive flavin 4a-hydroperoxide intermediate has allowed a detailed analysis of the reaction with oxygen. In the model of this reaction intermediate, fitted to the active site of p-hydroxybenzoate hydroxylase, all possible positions of the distal oxygen were surveyed by rotating this oxygen about the single bond between the C4a and the proximal oxygen. It was found that the distal oxygen is free to sweep an arc of about 180 degrees in the active site. The flavin 4a-peroxide anion, which is formed after reaction of molecular oxygen with reduced FAD, might accept a proton from an active-site water molecule or from the hydroxyl group of the substrate. The position of the oxygen to be transferred with respect to the substrate appears to be almost ideal for nucleophilic attack of the substrate onto this oxygen. The oxygen is situated above the 3-position of the substrate where the substitution takes place, at an angle of about 60 degrees with the aromatic plane, allowing strong interactions with the pi electrons of the substrate. Polarization of the peroxide oxygen-oxygen bond by the enzyme may enhance the reactivity of flavin 4a-peroxide.     

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.     

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.     

Mechanistic studies of p-hydroxybenzoate hydroxylase reconstituted with 2-Thio-FAD

2-Thio-FAD (oxygen substituent at position 2 is replaced by sulfur) was used to reconstitute the apoenzyme of p-hydroxybenzoate hydroxylase. The 2-thio-FAD enzyme differs from native enzyme in several respects. While the native enzyme catalyzes the fully coupled hydroxylation of p-hydroxybenzoate, the 2-thio-FAD enzyme shows no hydroxylation of this substrate, instead reducing molecular oxygen to hydrogen peroxide. The rate of reduction of 2-thio-FAD p-hydroxybenzoate hydroxylase by NADPH in the presence of substrate was 7-fold faster than with the native enzyme. However, the oxygen reactivity of the reduced 2-thio-FAD enzyme was less than 1% that of native enzyme. This slow oxygen reaction results in the very high KmO2 observed in steady state kinetic studies of the modified enzyme. Stopped flow studies of the oxygen reaction of the reduced 2-thio-FAD enzyme in the presence of substrate confirmed the formation of a transient intermediate. The spectrum of this intermediate is very similar to those of the flavin-C(4a) adducts obtained with 2-thio-FMN lactate oxidase. This evidence suggests that reduced 2-thio-FAD p-hydroxybenzoate hydroxylase forms a flavin-C(4a)-hydroperoxide on reaction with oxygen in a reaction analogous to that with native enzyme, but that the resulting peroxyflavin is incompetent as an oxygenating species, breaking down instead to oxidized 2-thio-FAD enzyme and hydrogen peroxide.     

Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism

Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.     

Using Raman spectroscopy to monitor the solvent-exposed and "buried" forms of flavin in p-hydroxybenzoate hydroxylase

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).     

pH-induced changes in activity and conformation of NADH oxidase from Thermus thermophilus

Thermus thermophilus NADH oxidase (NOX) activity exhibits a bell-shaped pH-dependency with the maximal rate at pH 5.2 and marked inhibition at lower pH. The first pH transition, from pH 7.2 to pH 5.2, results in more than a 2-fold activity increase with protonation of a group with pKa=6.1+/-0.1. The difference in fluorescence of the free and enzyme-bound flavin strongly indicates that the increase in enzyme activity in a pH-dependent manner is related to a protein-cofactor interaction. Only one amino acid residue, His75, has an intrinsic pKa approximately 6.0 and is localized in proximity (<10 A) to N5-N10 of the isoalloxazine ring and, therefore, is able to participate in such an interaction. Solvent acidification leads to the second pH transition from pH 5.2 to 2.0 that results in complete inhibition of the enzyme with protonation of a group with an apparent pKa=4.0+/-0.1. Inactivation of NOX activity at low pH is not caused by large conformational changes in the quaternary structure as judged by intrinsic viscosity and sedimentation velocity experiments. NOX exists as a dimer even as an apoprotein at acidic conditions. There is a strong coupling between the fluorescence of the enzyme-bound flavin and the intrinsic tryptophans, as demonstrated by energy transfer between Trp47 and the isoalloxazine ring of flavin adenine dinucleotide (FAD). The pH-induced changes in intrinsic tryptophan and FAD fluorescence indicate that inhibition of the FAD-binding enzyme at low pH is related to dissociation of the flavin cofactor, due to protonation of its adenine moiety.