Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase.

Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications for NADPH recognition and structural stability.

Phe161 and Arg166 of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens belong to a newly discovered sequence motif in flavoprotein hydroxylases with a putative dual function in FAD and NADPH binding [1]. To study their role in more detail, Phe161 and Arg166 were selectively changed by site-directed mutagenesis. F161A and F161G are catalytically competent enzymes having a rather poor affinity for NADPH. The catalytic properties of R166K are similar to those of the native enzyme. R166S and R166E show impaired NADPH binding and R166E has lost the ability to bind FAD. The crystal structure of substrate complexed F161A at 2.2 A is indistinguishable from the native enzyme, except for small changes at the site of mutation. The crystal structure of substrate complexed R166S at 2.0 A revealed that Arg166 is important for providing an intimate contact between the FAD binding domain and a long excursion of the substrate binding domain. It is proposed that this interaction is essential for structural stability and for the recognition of the pyrophosphate moiety of NADPH.

Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants.

The conserved residues His-162 and Arg-269 of the flavoprotein p-hydroxybenzoate hydroxylase (EC 1.14.13.2) are located at the entrance of the interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis. The catalytic properties of H162R, H162Y, and R269K were similar to the wild-type enzyme. However, less conservative His-162 and Arg-269 replacements strongly impaired NADPH binding without affecting the conformation of the flavin ring and the efficiency of substrate hydroxylation. The crystal structures of H162R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 A resolution, respectively. Both structures are virtually indistinguishable from the wild-type enzyme-substrate complex except for the substituted side chains. In contrast to wild-type p-hydroxybenzoate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate. NADPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyrocarbonate inactivation, suggesting that His-162 is involved in NADPH binding. Based on these results and GRID calculations we propose that the side chains of His-162 and Arg-269 interact with the pyrophosphate moiety of NADPH. An interdomain binding mode for NADPH is proposed which takes a novel sequence motif (Eppink, M. H. M., Schreuder, H. A., and van Berkel, W. J. H. (1997) Protein Sci. 6, 2454-2458) into account.

Inhibition of human neutrophil elastase. 4. Design, synthesis, X-ray crystallographic analysis, and structure-activity relationships for a series of P2-modified, orally active peptidyl pentafluoroethyl ketones.

A series of P2-modified, orally active peptidic inhibitors of human neutrophil elastase (HNE) are reported. These pentafluoroethyl ketone-based inhibitors were designed using pentafluoroethyl ketone 1 as a model. Rational structural modifications were made at the P3, P2, and activating group (AG) portions of 1 based on structure-activity relationships (SAR) developed from in vitro (measured Ki) data and information provided by modeling studies that docked inhibitor 1 into the active site of HNE. The modeling-based design was corroborated with X-ray crystallographic analysis of the complex between 1 and porcine pancreatic elastase (PPE) and subsequently the complex between 1 and HNE.

Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding.

The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.

X-ray structure of antistasin at 1.9 A resolution and its modelled complex with blood coagulation factor Xa.

The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 A resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

Molecular design and characterization of an alpha-thrombin inhibitor containing a novel P1 moiety.

An inhibitor of alpha-thrombin was designed on the basis of the X-ray crystal structures of thrombin and trypsin. The design strategy employed the geometric and electrostatic differences between the specificity pockets of the two enzymes. These differences arise due to the replacement of Ser 190 in trypsin by Ala 190 in thrombin. The new inhibitor contained a tryptophan side chain instead of the arginine side chain that is present in the prototypical thrombin inhibitors. This inhibitor had a Ki value of 0.25 microM, displayed more than 400-fold specificity for thrombin over trypsin, and doubled the rat plasma APTT at a concentration of 44.9 microM. The X-ray crystal structure of the inhibitor/alpha-thrombin complex was determined. This represents the first reported three-dimensional structure of a thrombin/ inhibitor complex where the specificity pocket of the enzyme is occupied by a chemical moiety other than a guanidino or an amidino group. As was predicted by the molecular model, the tryptophan side chain docks into the specificity pocket of the enzyme. This finding is in contrast with the indole binding region of thrombin reported earlier [Berliner, L. J., & Shen, Y. Y. L. (1977) Biochemistry 16, 4622-4626]. The lower binding affinity of the new inhibitor for trypsin, compared to that for thrombin, appears to be due to (i) the extra energy required to deform the smaller specificity pocket of trypsin to accommodate the bulky indole group and (ii) the favorable electrostatic interactions of the indole group with the more hydrophobic specificity pocket of thrombin. The neutral indole group may be of pharmacological significance because the severe hypotension and respiratory distress observed following the administration of some thrombin inhibitors have been linked to the positively charged guanidino or amidino functionalities.

Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding.

A novel conserved sequence motif has been located among the flavoprotein hydroxylases. Based on the crystal structure and site-directed mutagenesis studies of p-hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescens, this amino acid fingerprint sequence is proposed to play a dual function in both FAD and NAD(P)H binding. In PHBH, the novel sequence motif (residues 153-166) includes strand A4 and the N-terminal part of helix H7. The conserved amino acids Asp 159, Gly 160, and Arg 166 are necessary for maintaining the structure. The backbone oxygen of Cys 158 and backbone nitrogens of Gly 160 and Phe 161 interact indirectly with the pyrophosphate moiety of FAD, whereas it is known from mutagenesis studies that the side chain of the moderately conserved His 162 is involved in NADPH binding.

Chitinase and beta-1,3-glucanase in the lutoid-body fraction of Hevea latex.

The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are, besides the chitin-binding protein hevein, its precursor and the C-terminal fragment of this precursor, proteins with enzymic activities: three hevamine components, which are basic, vacuolar, chitinases with lysozyme activity, and a beta-1,3-glucanase. Lutoid-body fractions from three rubber-tree clones differed in their contents of these enzyme proteins. The hevamine components and glucanase were isolated and several enzymic and structural properties were investigated. These enzymes are basic proteins and cause coagulation of the negatively charged rubber particles. The coagulation occurs in a rather narrow range of ratios of added protein to rubber particles, which indicates that charg neutralization is the determining factor. Differences in coagulation of rubber particles by lutoid-body fractions from various rubber clones can be explained by their content of hevamine and glucanase. Glucanase from the lutoid-body fraction may dissolve callus tissue and this may explain the observation that rubber-tree clones with a high glucanase content in this fraction produce more latex than clones with little glucanase. Sequence studies of two CNBr peptides of the glucanase indicate that this protein is homologous with glucanases from other plants, and that a C-terminal peptide, possibly involved in vacuolar targeting, may have been cleaved off.

Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding.

Arg44, located at the si-face side of the flavin ring in 4-hydroxybenzoate hydroxylase, was changed to lysine by site-specific mutagenesis. Crystals of [R44K]4-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of [R44K]4-hydroxybenzoate hydroxylase is identical to the wild-type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180 degrees in 50% of the molecules. The phi, psi angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4-Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type. [R44K]4-Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped-flow kinetics showed that the effector role of 4-hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.

Refined crystal structure of the interleukin-1 receptor antagonist. Presence of a disulfide link and a cis-proline.

Interleukin-1 (IL-1) molecules are cytokines involved in the acute-phase response against infection and injury. Three naturally occurring IL-1 molecules are known, two agonists: IL-1 alpha and IL-1 beta, and one antagonist, the IL-1 receptor antagonist (IL-1ra). Although IL-1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL-1ra has been solved at 0.21-nm resolution by molecular replacement using the IL-1 beta structure as a search model. The crystals contain two independent IL-1ra molecules which are very similar. IL-1ra has the same fold as IL-1 alpha and IL-1 beta. The fold consists of twelve beta-strands which form a six-stranded beta-barrel, closed on one side by three beta-hairpin loops. Cys69 and Cys116 are linked via a disulfide bond and Pro53 has been built in the cis-conformation. Comparison of the IL-1ra structure with the IL-1 alpha and IL-1 beta structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N-terminus up to the beginning of strand beta 1 and the loops D and G, is very different in the three IL-1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL-1 receptor.

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.

Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate. Evidence for a proton channel and a new binding mode of the flavin ring.

The crystal structures of wild-type p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens, complexed with the substrate analogues 4-aminobenzoate, 2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate have been determined at 2.3-, 2.5-, and 2.8-A resolution, respectively. In addition, the crystal structure of a Tyr222Ala mutant, complexed with 2-hydroxy-4-aminobenzoate, has been determined at 2.7-A resolution. The structures have been refined to R factors between 14.5% and 15.8% for data between 8.0 A and the high-resolution limit. The differences between these complexes and the wild-type enzyme-substrate complex are all concentrated in the active site region. Binding of substrate analogues bearing a 4-amino group (4-aminobenzoate and 2-hydroxy-4-aminobenzoate) leads to binding of a water molecule next to the active site Tyr385. As a result, a continuous hydrogen-bonding network is present between the 4-amino group of the substrate analogue and the side chain of His72. It is likely that this hydrogen-bonding network is transiently present during normal catalysis, where it may or may not function as a proton channel assisting the deprotonation of the 4-hydroxyl group of the normal substrate upon binding to the active site. Binding of substrate analogues bearing a hydroxyl group at the 2-position (2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate) leads to displacement of the flavin ring from the active site. The flavin is no longer in the active site (the "in" conformation) but is in the cleft leading to the active site instead (the "out" conformation). It is proposed that movement of the FAD out of the active site may provide an entrance for the substrate to enter the active site and an exit for the product to leave.

Crystal structures of mutant Pseudomonas aeruginosa p-hydroxybenzoate hydroxylases: the Tyr201Phe, Tyr385Phe, and Asn300Asp variants.

Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Tyr385Phe, and Asn300Asp, each complexed with the substrate p-OHB have been determined by X-ray crystallography. Crystals of these three mutants of the Pseudomonas aeruginosa enzyme, which differs from the wild-type Pseudomonas fluorescens enzyme at two surface positions (228 and 249), were isomorphous with crystals of the wild-type P. fluorescens enzyme, allowing the mutant structures to be determined by model building and refinement, starting from the coordinates for the oxidized P. fluorescens PHBH-3,4-diOHB complex [Schreuder, H.A., van der Laan, J.M., Hol, W.G.J., & Drenth, J. (1988) J. Mol. Biol. 199, 637-648]. The R factors for the structures described here are: Tyr385Phe, 0.178 for data from 40.0 to 2.1 A; Tyr201Phe, 0.203 for data from 40.0 to 2.3 A; and Asn300Asp, 0.193 for data from 40.0 to 2.3 A. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B.A., Ballou, D.P., & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding interactions of the tyrosine residues but caused no other changes in the enzyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered in the Tyr385Phe and Tyr201Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interactions of the carboxamide group with the flavin O(2) atom, and the alpha-helix H10 that begins at residue 297 is displaced, altering its dipole interactions with the flavin ring. The functional consequences of these changes in the enzyme structure and of the introduction of the carboxyl group at 300 are described and discussed in the accompanying paper (Palfey et al., 1994b).

The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions.

Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule, whereas in ovalbumin, a nonfunctional serpin, the reactive site loop is completely exposed and in an alpha-helical conformation. However, in neither conformation can the reactive site loop bind to target proteinases. Here we report the structure of an intact and cleaved human antithrombin complex. The intact reactive site loop is in a novel conformation that seems well suited for interaction with proteinases such as thrombin and blood coagulation factor Xa.

Formation of the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase by a disorder-order transition from the unactivated to the activated form.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) catalyzes the key first step in photosynthetic CO2 fixation, the reaction that incorporates CO2 into sugar. In this study, refined crystal structures of unactivated tobacco RuBisCO and activated RuBisCO from spinach and tobacco, in complex with the reaction-intermediate analog 2-carboxyarabinitol 1,5-bisphosphate (CABP), are compared. Both plant enzymes are hexadecameric complexes of eight large and eight small subunits with a total relative molecular mass of approximately 550,000. The comparison of activated and unactivated forms of RuBisCO provides insight into the dynamics of action of this enzyme. The catalytic site, which is open to the solvent in the unactivated enzyme, becomes shielded in the activated CABP complex. This shielding is accomplished by a 12-A movement of the active-site "loop 6" (residues 331-338) and a disorder-order transition of three loops near the active-site entrance, the N terminus, the C terminus, and a loop comprising residues 64-68. All these residues belong to the catalytic large subunit. Domain rotations of about 2 degrees are observed, also tightening the active-site cleft. These observations provide an explanation for the extremely tight binding (Kd < or = 10(-11) M) of the CABP molecule. A striking correlation exists between crystallographic temperature factors in the activated enzyme and the magnitude of the atomic movement upon activation.

Crystal structure of activated tobacco rubisco complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate.

The crystal structure of activated tobacco rubisco, complexed with the reaction-intermediate analogue 2-carboxy-arabinitol 1,5-bisphosphate (CABP) has been determined by molecular replacement, using the structure of activated spinach rubisco (Knight, S., Andersson, I., & Brändén, C.-I., 1990, J. Mol. Biol. 215, 113-160) as a model. The R-factor after refinement is 21.0% for 57,855 reflections between 9.0 and 2.7 A resolution. The local fourfold axis of the rubisco hexadecamer coincides with a crystallographic twofold axis. The result is that the asymmetric unit of the crystals contains half of the L8S8 complex (molecular mass 280 kDa in the asymmetric unit). The activated form of tobacco rubisco is very similar to the activated form of spinach rubisco. The root mean square difference is 0.4 A for 587 equivalent C alpha atoms. Analysis of mutations between tobacco and spinach rubisco revealed that the vast majority of mutations concerned exposed residues. Only 7 buried residues were found to be mutated versus 54 residues at or near the surface of the protein. The crystal structure suggests that the Cys 247-Cys 247 and Cys 449-Cys 459 pairs are linked via disulfide bridges. This pattern of disulfide links differ from the pattern of disulfide links observed in crystals of unactivated tobacco rubisco (Curmi, P.M.G., et al., 1992, J. Biol. Chem. 267, 16980-16989) and is similar to the pattern observed for activated spinach tobacco.

A continuous in vitro method for estimation of the bioavailability of minerals and trace elements in foods: application to breads varying in phytic acid content.

A continuous in vitro method for the estimation of the bioavailability of minerals and trace elements is presented. This in vitro method is believed to be more representative of in vivo physiological conditions than in vitro methods based on equilibrium dialysis, because dialysable components are continuously removed from the pancreatic digestion mixture. The continuous in vitro method is compared with the equilibrium in vitro method with respect to the dialysability of Ca, Mg, Fe, Cu and Zn from eight different types of bread (varying in phytic acid content). The results show a pronounced effect of continuous removal of dialysable components from the pancreatic digestion mixture on the dialysability of minerals and trace elements. Furthermore, removal of dialysable components influences the effect of phytic acid on the bioavailability of minerals and trace elements. For these two reasons the importance of removal of dialysable components in vitro for the estimation of bioavailability in vivo needs further investigation. The bioavailability of minerals and trace elements from bread samples is not related to the phytic acid content only. Therefore, the effect of phytic acid on the bioavailability of minerals and trace elements cannot be studied separately from the effects of other components on bioavailability.

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.

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.