Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyses a reaction in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (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 protein and isoalloxazine ring during catalysis. Earlier research showed that reduction of FAD occurs when the isoalloxazine of the FAD moves to the surface of the protein to allow hydride transfer from NADPH. This move is coordinated with protein rearrangements that are triggered by deprotonation of buried p-hydroxybenzoate through a H-bond network that leads to the surface of the protein. In this paper, we examine the involvement of this same H-bond network in the oxygen reactions-the initial formation of a flavin-C4a-hydroperoxide from the reaction between oxygen and reduced flavin, the electrophilic attack of the hydroperoxide upon the substrate to form product, and the elimination of water from the flavin-C4a-hydroxide to form oxidized enzyme in association with product release. These reactions were measured through absorbance and fluorescence changes in the FAD during the reactions. Results were collected over a range of pH for the reactions of wild-type enzyme and a series of mutant enzymes with the natural substrate and substrate analogues. We discovered that the rate of formation of the flavin hydroperoxide is not influenced by pH change, which indicates that the proton required for this reaction does not come from the H-bond network. The rate of the hydroxylation reaction increases with pH in a manner consistent with a pK(a) of 7.1. We conclude that the H-bond network abstracts the phenolic proton from p-hydroxybenzoate in the transition state of oxygen transfer. The rate of formation of oxidized enzyme increases with pH in a manner consistent with a pK(a) of 7.1, indicating the involvement of the H-bond network. We conclude that product deprotonation enhances the rate of a specific conformational change required for both product release and the elimination of water from C4a-OH-FAD.     

Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins

Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.     

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.     

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.     

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.     

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.     

Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.

The crystal structure of the reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with its substrate p-hydroxybenzoate, has been obtained by protein X-ray crystallography. Crystals of the reduced form were prepared by soaking crystals of the oxidized enzyme-substrate complex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-bound FAD by NADPH. This was confirmed by single crystal spectroscopy. X-ray data to 2.3 A were collected on oscillation films using a rotating anode generator as an X-ray source. After data processing and reduction, restrained least squares refinement using the 1.9 A structure of the oxidized enzyme-substrate complex as a starting model, yielded a crystallographic R-factor of 14.8% for 11,394 reflections. The final model of the reduced complex contains 3,098 protein atoms, the FAD molecule, the substrate p-hydroxybenzoate and 322 solvent molecules. The structures of the oxidized and reduced forms of the enzyme-substrate complex were found to be very similar. The root-mean-square discrepancy for all atoms between both structures was 0.38 A. The flavin ring is almost completely planar in the final model, although it was allowed to bend or twist during refinement. The observed angle between the benzene and the pyrimidine ring is 2 degrees. This value should be compared with observed values of 10 degrees for the oxidized enzyme-substrate complex and 19 degrees for the enzyme-product complex. The position of the substrate is virtually unaltered with respect to its position in the oxidized enzyme. No trace of a bound NADP+ or NADPH molecule was found.     

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.     

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).     

Oxidation-reduction potential studies on p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The oxidation-reduction potential of p-hydroxybenzoate hydroxylase (4-hydroxybenzoate, NADPH: oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) from Pseudomonas fluorescens has been measured in the presence and absence of p-hydroxybenzoate using spectrocoulometry. The native enzyme demonstrated a two-electron midpoint potential of -129 mV during the initial reductive titration. The midpoint potential observed during subsequent oxidative and reductive titrations was -152 mV. This marked hysteresis is proposed to arise from the oxidation and reduction of the known air-sensitive thiol group on the enzyme (Van Berkel, W.J.H. and Müller, F. (1987) Eur. J. Biochem. 167, 35-46). Redox titrations of the enzyme in the presence of substrate showed a two-electron midpoint potential of -177 mV. No spectral or electrochemical evidence for the thermodynamic stabilization of any flavin semiquinone was observed in the titrations performed. These data show that the affinity of the apoenzyme for the hydroquinone form of FAD is 150-fold greater than for the oxidized flavin and that the substrate is bound to the reduced enzyme with a 3-fold lower affinity than to the oxidized enzyme. These data are consistent with the view that the stimulatory effect of substrate binding on the rate of enzyme reduction by NADPH is due to the respective geometries of the bound FAD and NADPH rather than to a large perturbation of the oxidation-reduction potential of the bound flavin coenzyme.     

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.     

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).     

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.     

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.     

NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution

NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.     

Structural studies on flavin reductase PheA2 reveal binding of NAD in an unusual folded conformation and support novel mechanism of action

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 A), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 A). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K(d) = 10 nm) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis.     

Mechanistic insights into p-hydroxybenzoate hydroxylase from studies of the mutant Ser212Ala.

In the crystal structure of native p-hydroxybenzoate hydroxylase, Ser212 is within hydrogen bonding distance (2.7 A) of one of the carboxylic oxygens of p-hydroxybenzoate. In this study, we have mutated residue 212 to alanine to study the importance of the serine hydrogen bond to enzyme function. Comparisons between mutant and wild type (WT) enzymes with the natural substrate p-hydroxybenzoate showed that this residue contributes to substrate binding. The dissociation constant for this substrate is 1 order of magnitude higher than that of WT, but the catalytic process is otherwise unchanged. When the alternate substrate, 2,4-dihydroxybenzoate, is used, two products are formed (2,3,4-trihydroxybenzoate and 2,4, 5-trihydroxybenzoate), which demonstrates that this substrate can be bound in two orientations. Kinetic studies provide evidence that the intermediate with a high extinction coefficient previously observed in the oxidative half-reaction of the WT enzyme with this substrate is composed of contributions from both the dienone form of the product and the C4a-hydroxyflavin. During the reduction of the enzyme-2,4-dihydroxybenzoate complex by NADPH with 2, 4-dihydroxybenzoate, a rapid transient increase in flavin absorbance is observed prior to hydride transfer from NADPH to FAD. This is direct evidence for movement of the flavin before reduction occurs.     

Crystal structure of the flavin reductase component (HpaC) of 4-hydroxyphenylacetate 3-monooxygenase from Thermus thermophilus HB8: Structural basis for the flavin affinity

The two-component enzyme, 4-hydroxyphenylacetate 3-monooxygenase, catalyzes the conversion of 4-hydroxyphenylacetate to 3,4-dihydroxyphenylacetate. In the overall reaction, the oxygenase component (HpaB) introduces a hydroxyl group into the benzene ring of 4-hydroxyphenylacetate using molecular oxygen and reduced flavin, while the reductase component (HpaC) provides free reduced flavins for HpaB. The crystal structures of HpaC from Thermus thermophilus HB8 in the ligand-free form, the FAD-containing form, and the ternary complex with FAD and NAD(+) were determined. In the ligand-free form, two large grooves are present at the dimer interface, and are occupied by water molecules. A structural analysis of HpaC containing FAD revealed that FAD has a low occupancy, indicating that it is not tightly bound to HpaC. This was further confirmed in flavin dissociation experiments, showing that FAD can be released from HpaC. The structure of the ternary complex revealed that FAD and NAD(+) are bound in the groove in the extended and folded conformation, respectively. The nicotinamide ring of NAD(+) is sandwiched between the adenine ring of NAD(+) and the isoalloxazine ring of FAD. The distance between N5 of the isoalloxazine ring and C4 of the nicotinamide ring is about 3.3 A, sufficient to permit hydride transfer. The structures of these three states are essentially identical, however, the side chains of several residues show small conformational changes, indicating an induced fit upon binding of NADH. Inactivity with respect to NADPH can be explained as instability of the binding of NADPH with the negatively charged 2'-phosphate group buried inside the complex, as well as a possible repulsive effect by the dipole of helix alpha1. A comparison of the binding mode of FAD with that in PheA2 from Bacillus thermoglucosidasius A7, which contains FAD as a prosthetic group, reveals remarkable conformational differences in a less conserved loop region (Gly83-Gly94) involved in the binding of the AMP moiety of FAD. These data suggest that variations in the affinities for FAD in the reductases of the two-component flavin-diffusible monooxygenase family may be attributed to difference in the interaction between the AMP moiety of FAD and the less conserved loop region which possibly shows structural divergence.     

Involvement of the flavin si-face tyrosine on the structure and function of ferredoxin-NADP+ reductases

In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)