PROTEINS: Structure, Function, and Genetics 14178-190 (1992)

Crystal Structure of the Reduced Form of p-Hydroxybenzoate Hydroxylase Refined at 2.3 A Resolution Herman A. Schreuder, Jan M. van der Laan, Myra B.A. Swarte, Kor H. Kalk, Wim G.J. Hol, and Jan Drenth BIOSON Research Institute, University of Groningen, 9747 AG Groningen, The Netherlands The crystal structure of the ABSTRACT reduced form of the enzyme p-hydroxybenzoate hydroxylase from Pseudomonas tluorescem, 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-substratecomplex in deaerated mother liquor containing 300-400 mM NADPH. A rapid bleaching of the crystals indicated the reduction of the enzyme-boundFAD 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 enzymesubstrate 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-hydroxybenzoateand 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".This value should be compared with observed values of 10" for the oxidized enzyme-substrate complex and 19"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. 1992 Wiley-Liss, Inc.

tion-reduction reactions,' but also reactions involving covalent adducts of the flavin ring. Examples are lactate oxidase where a glycollate molecule, covalently linked to the N5 of the flavin ring, is a reaction intermediate2 and enzymes like p-hydroxybenzoate hydroxylase and bacterial luciferase where a f lavin 4a-hydroperoxide intermediate occurs during the r e a ~ t i o n . ~ , ~ Flavin has three redox states: the fully reduced form, the one electron reduced flavosemiquinone radical and the oxidized form. The redox potentials associated with the redox transitions can vary from +80 mV for the transition from oxidized flavin to the semiquinone form in thiamine dehydrogenase, to -495 mV for the transition from the semiquinone form to fully reduced flavin in A. Vinelandii flavodoxin.' The flavin reacts readily with oxygen in some enzymes, while in others it does not react at all. It is clear that the properties of the flavin ring are very much dependent on the protein it is associated with and extensive studies are being conducted in order to find factors by which the protein is able to modulate the properties of the bound flavin. After reports by several investigator^^-^ that certain reduced flavin model compounds posses a strongly bent flavin ring (-30" t o -35"), there has been speculation about the influence of the bending of the flavin ring on its properties.'.' For a class of one-electron transfer proteins, the flavodoxins, high resolution structures of different flavodoxins in different redox states have been obtained by X-ray crystallography. The flavin ring in the various structures and oxidation states was virtually planar and no correlation between bending These angle and redox potential is

Key words: flavoenzymes, monooxygenase, FAD, reduced flavin, flavin planarity, Pseudomonas flmrescens

Received August 15, 1991; revision accepted January 21, 1992. Address reprint requests to Herman Schreuder, BIOSON Research Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Abbreviations used: enzyme: p-hydroxybenzoate hydroxylase; E P enzyme-product; ES: enzyme-substrate; ES-ox: oxidized ES; ES-red: reduced ES; PHBH: p-hydroxybenzoate hydroxylase; product: 3,4-dihydroxybenzoat; substrate: p-hydroxybenzoate.

INTRODUCTION Flavoproteins are able to catalyze a wide variety of reactions including one- and two-electron oxida0

1992 WILEY-LISS, INC.

CRYSTAL STRUCTURE OF REDUCED PHBH

o

\.o

/

PHBH FAD pOHB

PHBH FA0 3.4 di OHB 4

k

NADPH.H"

PHBH FA0 pOHB NAOPH

/

0

Fig. 1. Catalytic cycle of p-hydroxybenzoate hydroxylase. PHBH: p-hydroxybenzoate hydroxylase; pOHB: p-hydroxybenzoate, the substrate; FADH,-0,: flavin 4a-hydroperoxide intermediate; 3,4-diOHB: 3,4-dihydroxybenzoate,the reaction product.

results agree with results by Miiller and coworkers obtained with nuclear magnetic resonance techn i q u e ~ . ' ~However, .~~ a bending of the flavin of 20" has been reported in the 2.4 A structure of oxidized trimethylamine dehydr0gena~e.l~ The flavoprotein p-hydroxybenzoate hydroxylase from Pseudomonas f luorescens has been studied in our laboratory by X-ray crystallography. This enzyme catalyzes the conversion of the aromatic substrate p-hydroxybenzoate into 3,4-dihydroxybenzoate. The reaction also requires NADPH and molecular oxygen:

0 H O G < +

0, + NADPH

+ H'

+

0-

0

JF*,

HO

HO

0

C

+ H,O

+NADP+

0-

Entsch et al.3 have proposed a scheme for this reaction that is summarized in Figure 1. The catalytic cycle of p-hydroxybenzoate hydroxylase starts with binding of the substrate, followed by reduction of the enzyme-bound FAD by NADPH. Subsequently, the reduced flavin reacts with molecular oxygen to form a covalent flavin 4a-hydroperoxide intermediate. Since molecular oxygen has a triplet ground state, this reaction is spin-forbidden. Flavin is most likely able to circumvent this problem by its ability to participate both in one- and in two-electron oxidationreduction reactions. The flavin probably first donates one electron to the oxygen molecule and continues the reaction after spin inversion, as hap-

179

pens in model reactions.16-ls This scheme is further supported by the observation that the flavin analog 5-deazaflavin, only capable of two-electron oxidation-reduction reactions,lg is reduced by NADPH, but does not react with oxygen." The flavin 4a-hydroperoxide intermediate is thought to hydroxylate the substrate in p-hydroxybenzoate hydroxylase, resulting in the formation of the reaction product 3,4dihydroxybenzoate. The catalytic cycle is completed after release of the product. The crystal structure of the enzyme-substrate complex has initially been reported by Wierenga et aLZ1 It appeared to consist of three domains: The "FAD binding domain", the "substrate binding domain" and the "interface domain." The active site is situated a t the interface of all three domains. The enzyme-substrate complex has recently been refined to 1.9 A resolution." The structure of the enzymeproduct complex is kn0wn,"7'~ and model building studies have given a reasonable model for the flavin 4a-hydroperoxide intermediate.24s25 In the present study, we report on the structure determination of the enzyme-substrate complex with the flavin in the two-electron reduced state and compare this structure with the other known structures of p-hydroxybenzoate hydroxylase.

MATERIALS AND METHODS Soaking and Mounting Procedure A pure fraction of homogeneous PHBH dimers was prepared according to the procedure of van der LaanZ6and van Berkel and Miiller.27 The fraction was crystallized in the presence of the substrate phydroxybenzoate following the procedure developed by Wierenga.'6,28,29 Bright . yellow crystals of the enzyme-substrate complex with oxidized flavin were obtained in this way. The spacegroup is C222,. When these crystals are soaked in a solution containing 0.1 M potassium phosphate, pH 7.5, 40% saturated ammonium sulfate, 1 mM p-hydroxybenzoate, 0.02 mM FAD, 0.15 mM EDTA, 0.10 mM reduced glutathione, and 20 mM NADPH or dithionite, a rapid bleaching of the crystals is observed, indicating that the flavin is reduced by the NADPH or dithionite, respectively. In order to be able to study the reduced intermediate by X-ray diffraction, several precautions were taken to prevent reoxidation: Just before the soaking experiment, 20 mM p-hydroxybenzoate and 300-400 mM NADPH were added to the soaking liquor and the solution was thoroughly deoxygenated by repeatedly alternating between vacuum and nitrogen in a closed nitrogen ~ystern.'~ Soaking and mounting took place in a small glass vessel (diameter = 6.5 cm, height = 3 cm) that could be closed air tight. The vessel, which contained an X-ray capillary fixed inside, had been flushed with nitrogen for 24 hours. Deoxygenated soaking buffer containing NADPH was transferred into the X-ray capil-

180

H.A. SCHREUDER ET AL.

lary by means of a syringe with a long, sharp needle which was pushed through the rubber stopper by which the vessel was sealed. Inside the vessel a slight overpressure of nitrogen was maintained. After the preparation of a nitrogen atmosphere in the vessel, a crystallization capillary containing crystals of the oxidized enzyme-substrate complex was opened and completely filled with a column of deoxygenated soaking buffer lacking NADPH. Subsequently, the crystallization capillary was carefully pushed through a hole in the rubber stopper and brought in contact with the X-ray capillary. The whole setup was then carefully rotated such that the crystallization capillary was oriented upside down, and a crystal from the crystallization capillary could tumble gently into the X-ray capillary via a continuous column of deoxygenated mother liquor. The crystallization capillary was removed when the crystal was inside the X-ray capillary. The crystal was left in the NADPH containing liquid in the X-ray capillary for 12 hours while the vessel was kept under nitrogen. Then the mother liquor was removed and the crystal was oriented with a glass fiber. The neck of the X-ray capillary was filled with a small column of soaking liquor containing 100 mM sodium dithionite, in order to prevent diffusion of oxygen into the X-ray capillary. Any contact of this dithionite containing liquid with the crystal was strictly avoided in order to prevent diffusion of dithionite or its breakdown products into the crystal and to prevent possible pH changes. Finally the X-ray capillary was removed from the vessel and closed with araldite. The crystal was now ready for X-ray diffraction studies.

Preliminary Two-DimensionalStudies In order to establish suitable soaking conditions, zero order precession pictures of soaked reduced crystals were made and compared with those taken from native oxidized crystals. Two-dimensional difference Fouriers were calculated using phases obtained from a partially refined model of the enzyme-substrate complex (Wierenga and Prick, unpublished results). Only small differences between oxidized and reduced crystals were observed. Interestingly, when reduced crystals were reoxidized, either by soaking in oxygenated mother liquor, or by leakage of oxygen into the X-ray capillary, significant peaks were found in difference Fouriers, similar to those for crystals that had been soaked for a long time in mother liquor containing a high concentration of the product, 3,4-dihydroxybenzoate. During the reduction-reoxidization procedure, apparently one catalytic cycle of the enzyme is completed, resulting in product formation in the cryst a l ~Based . ~ ~on this knowledge, a precession picture was made of each reduced crystal after three-dimensional data collection in order to make sure that no reoxidation had taken place.

TABLE I. Completeness as a Function of Resolution for the Data Used in Refinement and Man Calculations ~

Resolution range (A) 6.00-4.24 4.24-3.46 3.46-3.00 3.00-2.68 2.68-2.45 2.45-2.30

~

~~~~

Completeness (%) 84.7 77.3 65.3 53.9 58.5 37.4

Data Collection Data were collected on film (CEA reflex251 using an Enraf-Nonius oscillation camera mounted on a n Elliot GX-6 rotating anode generator. Cu-Ku radiation was obtained with a Huber graphite monochromator. All films were scanned using a 50 pm raster on a Scandig microdensitometer (Joyce-Loeble) and processed with a modified version of the Munich oscillation ~ a c k a g e . ~ Refinement '.~~ of the cell-dimensions yielded: a=71.5 A, b=146.4 A, c=88.0 A, which are only slightly different from the native oxidized crystals: a = 71.7 A, b = 145.8 A, c = 88.2 A."' Film to film scaling was carried out by applying scale and isotropic temperature factors per film, calculated according to a n extension of the method of Hamilton et al.3zScaling and merging of the films was carried out using programs from the Groningen BIOMOL protein structure determination software package. From a total of 82,091 observations, 13,308 unique and fully recorded reflections were left after data reduction. The resulting dataset is 61% complete to 2.3 A and has a n internal R,, of 5.8%, where R,, = C II Fi I - I Fuu,iII E / F a u , iI and is the average of all measurements of the same reflection. The relatively low completeness of the data is of some concern, because it could lead to distortions of the electron density. Analysis of the completeness as a function of resolution (Table I) reveals that the low resolution shell from 6 to 4.24 A is resonably complete (84.7%) and that the completeness gradually decreases to 37.4% for the highest resolution shell. The distribution of missing reflections was examined on a n Evans & Sutherland computer graphics display, using the WHATIF program.33 A cone of data of about 10" is missing around the b* axis, which was parallel to the spindle axis during data collection. The remaining missing data is more or less evenly distributed and increases at higher resolution. These missing reflections are probably partials and weak reflections, that have been rejected during the data processing procedure. In order to compensate for differences in falloff of IF/' as a function of the scattering angle between the 2.5 A dataset recorded from the oxidized crystals of the enzyme-substrate complexz1 and the dataset

181

CRYSTAL STRUCTURE OF REDUCED PHBH

from the reduced crystals recorded in the present study, the datasets were brought to the same scale using six anisotropic B-factors with the program KBRANI from the Groningen BIOMOL protein structure determination package. An R-factor of 14.2% was found for 10,141 common reflections between both datasets after scaling.

Refinement It was known from preliminary refinement runs that the model of the E S complex with oxidized flavin could serve directly as a starting model for the refinement of the ES complex with reduced flavin. The cell-dimensions of the crystals of the oxidized enzyme-substrate complex and the reduced enzymesubstrate complex differ less than 0.5% and the initial R-factor of 27% for the oxidized starting model indicated a reasonable starting point for the refinement. Refinement was carried out using a highly optimized version of the Konnert-Hendrickson restrained least squares refinement program PROLSQ.34 This program was kindly provided by Dr. M.G. Rossmann and colleagues a t Purdue and adapted to our local environment by Dr. Anne Volbeda. It ran on a Cyber205 in Amsterdam and needs about 260 CPU seconds per cycle. The structure of the enzyme-substrate-complex (R= 15.6% at 1.9 A resolution) including 330 solvent molecules was used as a starting model. The great similarity between the oxidized and the reduced form of p-hydroxybenzoate hydroxylase is exemplified by the fact that a single cycle of Konnert-Hendrickson restrained least squares refinement reduced the Rfactor for data between 6.0 A and 3.0 A to 19.5%. Both positional and thermal parameters were refined i n this first cycle, yielding a n r.m.s. positional shift of 0.28 A, and a n r.m.s. B-factor shift of 2.2 A". 11 cycles of restrained least squares refinement with a resolution gradually increasing from 3.0 A to 2.3 A, reduced the R-factor to 16.3%. At this stage the model was inspected on an Evans & Sutherland PS390 computer graphics display running FRODO software.35 The model fitted well in the electron density and did not require rebuilding parts of the protein or FAD. However, due to either poor electron density, high B-factors or a position too close to protein atoms, 33 solvent atoms were removed, and 25 new solvent molecules were placed, based on a UA weighted F, - F, difference map.36 A final R-factor of 14.8% for 11,394 reflections between 6.0 A and 2.3 A was reached after 10 further cycles of refinement. The results are summarized in Table 11. The final model contains 322 bound solvent molecules. The mean coordinate error was estimated from a Luzzati plot37 to be -0.2 (Fig. 2). Although this value is the same as obtained for the 1.9 A structure of the oxidized enzyme-substrate complex, we can expect the accuracy of the present structure to be

TABLE 11. Final Parameters and Results of the Refinement of the Reduced ES Complex

Rt Distances Bond distances Angle distances Planar 1-4 distance Planes Chiral volumes Non-bonded contacts Single torsion contacts Multiple torsion contacts Possible hydrogen bonds Thermal parameter correlations Main chain bond Main chain angle Side chain bond Side chain angle

Model value* 14.8 0.012 0.030 0.032

Target

IT

0.153

0.020 0.030 0.040 0.020 0.150

0.196 0.245 0.257

0.350 0.350 0.350

0.011

4.806 5.263 8.388 10.889

5.0 5.0 7.0 10.0

*Model values are root-mean-square differences from ideal values (in A for distances, in PIz for thermal parameter correlations and in for chiral volumes). Target u is the inverse square root of the least squares weight used for the parameter listed as described by Hendrickson and K0nne1-t.~~

somewhat poorer because it is based on X-ray data of lower resolution (2.3 A). Also the low completeness of 61% of the data will lead to a less well defined structure. However, since the reduced model is based upon the 1.9 A ES-ox model, we expect the errors to be less than if the structure had been solved de novo. To verify that the observed small differences are real, we calculated and Fobs - Fca,c,oxdifference was based on the model of oximap, where Fcalc,ox dized PHBH, superimposed onto the final reduced model, The R-factor for data between 6.0 A and 3.0 A between Fobsand Fcalc,oxis 19%, indicating that a large part of the drop in R-factor in the first cycle of least squares refinement can be attributed to the 0.3 8, rigid body shift between oxidized and reduced PHBH. The difference map showed small positive and negative peaks that support the observed positional differences. Some examples, which pertain to differences discussed in the results section, are a 1.9 u peak at the si-side and a -2.5 u peak at the re-side of the flavin ring near the N1, supporting the small differences in this region. A +2.2 u peak indicates that the N10 is somewhat out of plane. A -2.2 u near the substrate 0 4 and -2.2 cr and +2.7 u peaks near the guanidinium group of Arg214 support the small movements seen in Figure 8.

+

Single Crystal Spectroscopy Microspectrophotometric measurements on single crystals were performed with a Zeiss UMSP I double

182

H.A.SCHREUDER ET AL.

0.40

0.35 30

0 301

80 25 $0

20

&O

15

0 10 0 05

O O O O 00

0 10

1/0

0 20 0 30 IANGSTROM**-l)

0 40

Fig. 2. R-factor as a function of resolution (thick line). Theoretical curves for mean coordinate errors of 0.1 A, 0.2 A, and 0.3 A3' are plotted in thin lines. The expected mean coordinate error, which can be derived from this curve is -0.2 A.

beam recording microspectrophotometer at the Biozentrum in Basel. Small, thin crystals (0.05*0.1*0.2 mm3) were soaked in NADPH containing mother liquor under a nitrogen atmosphere as described previously. A small quartz capillary was used (diameter = 0.7 mm) to "mount" the crystals. The diameter of the measuring area was about 32 pm and the light path through the crystals was about 0.05 mm. The absorbance of the solvent just next to the crystal was measured and subtracted from the absorbance measured when the light went through the crystal. Spectra were recorded using unpolarized light with the light beam parallel to the a axis of the crystals. In the presence of NADPH and p-hydroxybenzoate, which strongly absorb around 340 nm and around 280 nm respectively, spectra could only be recorded between 400 and 500 nm. After recording the spectrum in this wavelength range, the capillary was opened and the crystals were rinsed with aerated mother liquor lacking NADPH and p-hydroxybenzoate. After 1 hour, the crystal was mounted again in the quartz capillary and a full spectrum was recorded between 270 nm and 500 nm.

RESULTS AND DISCUSSION Spectra of p-Hydroxybenzoate Crystals Although the bleaching of the p-hydroxybenzoate hydroxylase crystals upon reduction was clearly visible, the changes in X-ray diffraction pattern were very small. To ascertain that the bound flavin was in the reduced state, single crystal spectroscopy was performed on p-hydroxybenzoate hydrozylase crystals. Because the crystals contain a very high protein concentration (about 500 mg/ml), the absorbance of the large crystals used for X-ray data collection was too high for optical spectroscopy. Therefore smaller crystals were used. Due to absorbance by NADPH and p-hydroxybenzoate, the spectrum of the reduced crystals could only be measured between 400 and 500 nm as mentioned above. Nevertheless, it is clear from the recorded spectra (Fig. 3), that the crystals soaked anaerobically with

NADPH lack the absorbtion peak at 450 nm indicative of oxidized flavin. From this we conclude that the reduced form of the flavin predominates in the crystals which were used for data collection. The spectra recorded after washing the crystals with aerated mother liquor without NADPH or phydroxybenzoate show a n absorption peak at 450 nm which indicates oxidized flavin. The similarity of spectra recorded for a crystal in a cylindrical capillary and between flat quartz plates, proves that the shape of the capillary does not influence the spectrum significantly. The A280/A450 ratio measured was in the range of 10-11, which is higher than the value of pure p-hydroxybenzoate hydroxylase in solution: 8-9. This discrepancy probably arises from anisotropy of crystal ab~orption.~' Due to the shape of the crystals, it was only possible to measure the absorbtion along the a axis. The long axis of the flavin ring in oxidized p-hydroxybenzoate hydroxylase makes an angle of approximately 24" with the a axis, while the electric dipole moment of the flavin ring makes a n angle of 15" with the long axis of the flavin ring.39s40The angle between the electric dipole moment and the a axis is approximately 14".Light with its electric field vector perpendicular to the electric dipole moment (as is true for a light beam parallel to the electric dipole moment) is hardly absorbed by oxidized flavin at 450 nm (see, e.g., Fig. 3 in ref. 391, explaining the low absorbtion at 450 nm for a light beam parallel to the a axis.

Atomic Coordinates The coordinates of the starting model of the phydroxybenzoate hydroxylase-substrate complex with oxidized flavin shifted only slightly away from their original position during the refinement. For all atoms, excluding bound solvent atoms, the rootmean-square shift is 0.38 h;. For the Ccx coordinates it is 0.28A. These values are of the same magnitude as, e.g., the root-mean-square differences of 0.47and 0.39 A observed between the coordinates of Bovine Trypsin Inhibitor in different crystal forms.41 Three crystal forms of this inhibitor were obtained and the structures have been refined to high resolution. The differences between these structures are greater than the expected coordinate error and give some hints about the possible intrinsic motions of proteins. The observed differences between the oxidized and reduced form of the p-hydroxybenzoate hydroxylasesubstrate complex are thus within the limits of normal intrinsic motion in protein molecules. Planarity of the Flavin Ring In view of the strongly bent reduced flavins found by Kierkegaard et al.,5-7 and the planar reduced flavins observed by Muller and coworker^,'^.'^ we were particularly interested in the planarity of the flavin ring in the reduced state. A view along the

CRYSTAL STRUCTURE OF REDUCED PHBH

183

4.0

I

3.5

.o

0.8

3.0 0.6

2.5

0.4

Lu U

z < m

m

2.0

0.2

0 Ln

m

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-hyd...
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