THE JOURNAL 0 1989 by The American Society for Biochemistry

OF BIOLOGICAL CHEMISTRY and Molecular Biology, Inc

Vol. 264. , No. 15, Issue of May 25, pp. 8791-8801,1989 Printed in U.S.A.

Binding of Isotopically Labeled Substrates, Inhibitors, and Cyanide by Protocatechuate 3,4-Dioxygenase*

(Received for publication, September 19,1988)

Allen M. Orville and John D. Lipscomb$ From the Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, Minnesota 55455

Binding of ligands to the active site Fe3+ of proto- catechuate 3,4-dioxygenase is investigated using EPR-detected transferred hyperfine coupling from isotopically labeled substrates, inhibitors, and cyanide. Broadening is observed in EPR resonances from the anaerobic enzyme complex with homoprotocatechuate (3,4-dihydroxyphenylacetate), a slow substrate, en- riched with "0 ( I = %) in either the 3-OH or the 4-OH group. This shows that this substrate binds directly to the Fe3+ and strongly suggests that an iron chelate can be formed. Cyanide is known to bind to the enzyme in at least two steps, forming first a high spin and then a low spin complex (Whittaker, J. W., and Lipscomb, J. D. (1984) J. Biol. Chen. 259, 4487-4495). Hyperfine broadening from [13C]cyanide ( I = Vi) is observed in the EPR spectra of both complexes, showing that cya- nide is an Fe3+ ligand in each case. Cyanide binding is also at least biphasic in the presence of protocatechuate (PCA). The initial high spin enzyme-PCA-cyanide com- plex forms rapidly and exhibits a unique EPR spec- trum. Broadening from PCA enriched with 170 in either the 3-OH or the 4-OH group is detected showing that PCA binds to the iron, probably as a chelate com- plex. In contrast, no broadening from [13C]cyanide is detected for this complex suggesting that cyanide binds at a site away from the Fe3+. Steady state kinetic measurements of cyanide inhibition of PCA turnover are consistent with two rapidly exchanging cyanide binding sites that inhibit PCA binding and which can be simultaneously occupied. Formation of the nearly irreversible, low spin enzyme-PCA-cyanide complex is competitively inhibited by PCA. Transient kinetics of the formation of this complex are second order in cy- anide implying that two cyanides bind. Broadening in the EPR spectrum of this complex is detected from ["Clcyanide, but not from [170]PCA, suggesting that PCA is displaced. This study provides the first direct evidence for chelation of the active site Fe3+ by sub- strates and for a small molecule binding site away from the iron in intradiol dioxygenases.

Protocatechuate 3,4-dioxygenase (EC 1.13.1.3) serves as a central enzyme in the biodegradation of aromatic compounds (1-3). It catalyzes reactions of the following type:

* This work was supported by Grant GM 24689 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ T o whom correspondence should be addressed 4-225 Millard Hall, Dept. of Biochemistry, University of Minnesota, Minneapolis, MN55455.

R R

SCHEME 3

The open chain product contains one oxygen atom from O2 in each of the terminal carboxyl functions. The enzyme has been isolated from several widely divergent bacteria (see, for example, Refs. 4-7). All of the known protocatechuate 3,4- dioxygenases contain essential Fe3+ that has provided a ver- satile spectroscopic probe for the structure of the active site and the mechanism of action of the enzyme (see, for example, Refs. 4,8). Although the quaternary structures of the enzymes vary significantly, the iron site appears to be remarkably well conserved. Other Fe3+-containing dioxygenases such as cate- chol 1,2-dioxygenase exhibit very similar spectral properties suggesting these enzymes use analogous mechanisms (9).

Spectroscopic studies suggest that the protein ligands to the active site iron are 2 tyrosines (see, for example, (10-12)) and 2 histidines (13).* Hyperfine coupling between [170]water and electronic spin of the Fe3+ leads to broadening of the EPR spectrum showing that water is also a ligand (14). The broad- ening is not observed in the spectra of substrate complexes suggesting that the water is displaced. In contrast, broadening is observed in spectra of enzyme complexes with monodentate ligands such as 4-HBA as well as substrate analogs that assume the ketonized configuration such as 2-hydroxyiso- nicotinic N-oxide (14, 15). Since 4-HBA is known from reso- nance Raman studies to bind directly to the iron (lo), at least two sites in the iron coordination must be accessible to exogenous ligands.

Based on these and related studies, a mechanism has been proposed in which deprotonated substrate hydroxyl groups initially occupy both of the iron sites available for exogenous ligands (14). Oxygen is then thought to attack the substrate at the 4-carbon, forcing it to become tetrahedral and promot- ing tautomerization of the substrate to the keto form. Release of the resulting keto oxygen from the iron is proposed to allow a change in the orientation of the substrate in the active site and subsequent coordination of the distal atom of the attack-

' The abbreviations used are: PCA, protocatechuic acid or 3,4- dihydroxybenzoic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; HPCA, homoprotocatechuic acid or 3,4-dihydroxyphenylacetic acid; 3-HBA, 3-hydroxybenzoic acid; 4-HBA, 4-hydroxybenzoic acid; 3-HPA, 3-hydroxyphenylacetic acid; 4-HPA, 4-hydroxyphenylacetic acid; PMSF, phenylmethylsulfonyl fluoride; T, tesla (=lO,OOO Gauss).

The recently completed x-ray crystal structure of protocatechuate 3,4-dioxygenase isolated from Pseudomonas aeruginosa shows that this ligand assignment is correct (33).

8791

8792 Ligand Binding by Protocatechuate 3,4-Dioxygenase

O N \:

A k R I

SCHEME 2

ing 0 2 to the iron site vacated. Oxygen-oxygen bond cleavage and insertion into the ring is then proposed to proceed from this peroxy-intermediate.

Iron chelation by PCA has proven difficult to test. Reso- nance h m a n studies, for example, have shown that PCA binds to the iron but do not give the coordination number (10). The best evidence for chelation has come from NMR experiments which have been interpreted in light of model complexes to show that the inhibitor 4-methyl catechol can form a chelate complex with the iron (16). Potentially, hyper- fine coupling from 170-enriched substrate could be used to directly detect chelation, but the EPR resonances from the anaerobic enzyme-PCA complex are unusually broad so that small additional hyperfine broadening is not readily detecta- ble. We have proposed that the broad resonances of the substrate complex reflect the ability of the substrate to assume many orientations in the active site (5), which is consistent with the change in substrate orientation envisioned in the mechanism described above. Enzyme complexes of alternate substrates also exhibit considerable heterogeneity. In some cases, however, one species dominates allowing isolated, nar- row EPR resonances to be observed. In the current study, one such substrate, HPCA, has been prepared with 170 incorpo- rated specifically into each oxygen. Investigation of the co- ordination of this and related inhibitor complexes to the iron strongly suggests that the chelated substrate analog complex is the predominant form.

The presence of multiple accessible iron ligand sites is also indicated by studies of cyanide binding by the enzyme. Native enzyme binds cyanide in at least a two-step process leading first to a high spin and then to a low spin complex. This is consistent with the successive binding of two cyanide ligands (14). If PCA is added to the initial cyanide complex a new high spin species is formed showing that cyanide and PCA bind simultaneously to the enzyme. In contrast to the broad EPR spectra of the substrate complexes, the spectrum of the enzyme-substrate-cyanide complex is sharp. The narrow EPR resonances potentially allow observation of hyperfine broad- ening from 170-labeled PCA and 13C-labeled cyanide so that binding can be directly assessed. The spectroscopic and ki- netic data presented here suggest that cyanide has binding sites both on and off the iron. In the initial enzyme-cyanide- PCA complex, cyanide appears to occupy only a remote site and PCA can coordinate to the active site iron via both OH groups. The fact that a small molecule binding at a remote site profoundly affects the substrate complex raises the pos- sibility that the site may have mechanistic significance as a binding site for oxygen or a substrate functional group.

MATERIALS AND METHODS AND RESULTS3

Substrate Ligation-Substrate ligation to the active site iron of protocatechuate 3,4-dioxygenase can, in principle, be

Portions of this paper (including “Materials and Methods” part of “Results”, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.

9.03 9.671 A

x 125

x 250 L

1 75 100 125 150

FIG. 1. EPR spectra of enzyme substrate complexes. Enzyme (97 FM) in 100 mM MOPS buffer, pH 7, was incubated anaerobically with either 10 mM PCA (A) or 10 mM HPCA (8) for 5 min prior to freezing by slow immersion in liquid N2. Samples prepared with 50 mM HPCA (not shown) had identical line shapes as that shown in B . The Kd of HPCA for the enzyme was determined by optical titration to be -20 p ~ . Spectra were recorded a t X-band under the following conditions: temperature, 7.5 K; modulation amplitude, 1.0 mT, mi- crowave power, 0.2 milliwatts. The g-values, field calibration, and relative gains are shown on the figure.

detected by observing transferred hyperfine broadening of EPR resonances due to 170-labeled PCA. Unfortunately, the EPR spectrum of the anaerobic enzyme complex with PCA (Fig. 1A) is inherently too broad to detect such broadening, which is typically only a few Gauss in magnitude. The enzyme complex with the “slow” substrate HPCA also has multiple EPR active species (Fig. 1B); however, the distribution of the species is markedly different. The majority component ac- counting for of the HPCA complex occurs as a species with E/D = 0.33 ( g = 9.68,4.28; E / D is reflective of electronic symmetry of the iron site, see Miniprint). This species has sufficiently narrow line width to allow detection of hyperfine broadening. As shown in Fig. 2 and summarized in Table I (see Miniprint), all of the resonances of the EPR spectrum of this species are broadened when *70 is incorporated specifi- cally into either the 3-OH or the 4-OH group of HPCA. Thus both the 3- and 4-OH groups can bind directly to the iron, probably simultaneously to form a chelate complex. Approx- imately 5% of the HPCA complex occurs as a species with E/D = 0.02 (g = 5.51, 6.52). The large inherent line width of the EPR resonances from this species makes the observation

The observed intensity of EPR resonances is strongly dependent on the isotropy of the spectrum. For example, ground state resonances

g = 6 region (E/D - 0) than in the g = 9.6 region (E/D - V3). See from S = 5/2 systems are approximately 60 times more intense in the

Ref. 5 for a discussion.

Ligand Binding by Protocatechuate 3,4-Dioxygenase 8793

150.0 152.5 155.0 157.5 H (mT)

FIG. 2. EPR spectra of "0-labeled HPCA-enzyme com- plexes. The g = 4.28 resonance of the spectrum shown in Fig. 1B is expanded to evaluate hyperfine broadening. Shown superimposed are spectra of anaerobic complexes with HPCA labeled in the 3-OH (long dashes) and the 4-OH (solid line), and unlabeled (short dashes). Protein samples were prepared and spectra were run under the conditions described in Fig. 1.

of broadening equivocal. However, it appears that incorpora- tion of I7O in the 4-OH broadens and decreases the intensity of the spectrum slightly (not shown). No broadening was observed in this region of the spectrum from the 3-[hydroszyl- I70]HPCA-enzyme complex. Thus this species may represent a monodentate Fe3+-substrate complex formed through the 4- OH group. No broadening is observed in any resonance when I7O is incorporated into the carboxyl group of HPCA. Hyper- fine broadening from "'0-enriched water is detectable in the EPR spectrum of native protocatechuate 3,4-dioxygenase due to direct ligation of water to the active site Fe3+ (14). No broadening from ['70]water is detected in any resonances after anaerobic addition of unlabeled HPCA suggesting that HPCA displaces the water.

Inhibitor Ligation-The enzyme complex with 4-HBA con- sists of two discrete species (EID = 0.32 and 0.24). The EPR spectrum of the enzyme complex with 4-[hydroxyl-I70]HBA shows broadening in at least one resonance from each species, showing that the hydroxyl group coordinates to the iron in each case (Fig. 3A and Table I). Since water binding to the iron has previously been demonstrated for each species (14), the data suggests that at least two sites for exogenous ligands are available in the iron coordination in each case. The analogous 3-[hydr0xyl-'~O]HBA complex consists of three species with EID = 0.33,0.25, and 0.18. The latter two species represent about 5% of the enzyme and are too broad to unequivocally detect broadening. The major species with E/D = 0.33 exhibits no broadening (Fig. 3B and Table I) in any resonance suggesting that the hydroxyl group is not coordinated to the iron. The EPR line shape of this species is similar to that of inhibitor-free enzyme. However, unlike free enzyme, the E / D = 0.33 species exhibits no broadening from [170]water.

The monohydroxyphenylacetate inhibitors 3-HPA and 4- HPA have lower affinities for the enzyme than do the mono- hydroxybenzoates. The enzyme complexes with 50 mM 3-

2

FIG. 3. EPR spectra of "0-labeled enzyme-monohydroxy- benzoate complexes. Enzyme (100 PM) in 100 mM MOPS buffer was mixed anaerobically with 50 mM 4-HBA ( A ) or 50 mM 3-HBA ( B ) and incubated for 5 min prior to freezing. The g = 4.3 region of the spectra of enzyme complexes with unenriched (-----) and 170H- enriched (-) complexes are shown superimposed. Spectra shown in B were indistinguishable. Conditions as in Fig. 1.

HPA or 4-HPA exhibit two EPR active species in approxi- mately equal concentrations. One of the species in each case is indistinguishable from native enzyme in terms of resonance position, width, and power saturation behavior as well as [I7O] water-induced hyperfine broadening. Moreover, the concen- tration of the species increases at lower inhibitor concentra- tion, thus it is likely that this species is free enzyme. EPR spectra of the enzyme complexes with 4-[hydr0xyl-'~O]HPA and 3-[hydr0xyl-'~O]HPA are broadened by hyperfine inter- actions (Table I) showing that the hydroxyl group is bound directly to the Fe3+ in each case. In contrast to the monohy- droxybenzoate complexes, [I70]water is bound to the Fe3+ in the enzyme.3-HPA complex but apparently not in the en- zyme. 4-HPA complex (Table I).

Spectral Studies of Cyanide Complex Formation-Previous studies (14) have shown that cyanide binds to the oxidized enzyme in at least two steps generating first a high spin (g = 7.84, 3.83, 1.73) and then a low spin species (g = 2.43, 2.21, 1.89). If PCA is added to the intermediate species, a new species with a unique EPR spectrum is formed (g = 4.41,4.23, 3.81, see inset of Fig. 6B) . The spectrum is markedly different than that of the anaerobic substrate complex and appears to result from a single species. The time course of optical changes caused by addition of PCA to the enzyme-cyanide complex are shown in Fig. 4 (top). The absorbance at 530 nm, which is characteristic of the high spin cyanide complex, shifts to 470 nm immediately after addition of PCA. Slowly, the re- sulting species converts to another species with an optical spectrum very similar to that observed for the low spin

8794

1.2

1.c

0.E

0.6

0.4

0 0.2 Q)

5 t o $ 1.0 0

-4 0.8

0.6

0.4

0.2

0

Ligand Binding by Protocatechuate 3,4-Dioxygenase

5 2.0

7 4.0 6 3.0

400 500 600 700 800 Wavelength (nm)

FIG. 4. Optical spectra of enzyme-cyanide complex forma- tion. Top, enzyme (54 FM) was mixed anaerobically with 140 mM NaCN ( E . I ) , in 400 mM MOPS buffer, pH 7.0, and 1.1 mM PCA was added (I3.S.Z). The time course of the reaction is illustrated by spectra recorded at 30-min intervals. The spectrum of the final complex was recorded after 16.5 h and was unchanged after 48 h. The spectrum of the enzyme ( E ) and the anaerobic enzyme-PCA ( E . S ) complex are shown for comparison. Temperature = 25 "C. Bottom, enzyme (60.5 FM) was mixed anaerobically with 278 mM NaCN ( E . I ) , in 500 mM MOPS buffer, pH 7.0, containing 100 mM Na2S04. PCA was added rapidly in the concentrations shown and the spectra scanned immediately. Temperature = 31 "C. All spectra in both panels have been normalized to correct for dilution (14%) on addition of NaCN.

complex observed after addition of cyanide alone. Since an isosbestic point is observed for this conversion a t 406 nm, it appears that either there are no intermediates or that the intermediates are very short lived. The optical spectrum of the semistable intermediate a t 470 nm is similar to that of the anaerobic substrate complex. However, the unique EPR spectrum of this complex suggests that it has a substantially different structure. As shown in Fig. 4 (bottom), the amount of the substrate complex-like, semistable intermediate formed at the start of the reaction is dependent on the concentration of PCA (and cyanide) present, suggesting that cyanide and PCA compete for a binding site on the enzyme. An apparent isosbestic point is observed a t 645 nm. However, the failure to form an isosbestic point a t -495 nm shows that the binding reaction is, in fact, complex. The order of addition of PCA and cyanide to the enzyme has no affect on the optical and EPR spectra of either the intermediate or the final complexes.

Isotopically labeled PCA and cyanide can be used to probe the iron coordination in the intermediates of the cyanide binding reaction. As shown in Fig. 5, A and B, hyperfine broadening from ["CC]cyanide is observed in both the high spin and low spin complexes formed during the reaction with cyanide alone. No narrowing of the spectrum is observed when ["Nlcyanide ( I = Yz) is used in place of [14N]cyanide ( I = 1). This shows that cyanide coordinates directly to the

H FIG. 5. EPR spectra of en~yme-['~C]cyanide complexes.

NaCN (200 mM) was mixed with enzyme (79 pM) in 200 mM MOPS buffer, pH 7.0, A , the sample was frozen immediately after mixing unenriched (-----) or 99% I3C-enriched (-) cyanide to trap the intermediate enzyme-cyanide complex. B , the sample from A was incubated at room temperature for 2 h to allow formation of the final enzyme-cyanide complex. Insets show the line shape of the complete EPR spectra (center = 210 mT, sweep = 400 mT). Spectra were measured under the conditions described in Fig. 1. The spectra in A are normalized to the same intensity ( W N complex spectrum X 1.26).

iron via the carbon in both complexes. If I70-labeled PCA is added anaerobically to the preformed high spin cyanide com- plex, hyperfine broadening is observed in the EPR spectra of the ternary enzyme complex no matter whether the isotope is present in the 3- or the 4-hydroxyl function (Fig. 6A). No broadening is observed in any resonance from this complex when it is formed with ['3C]cyanide (Fig. 6B). Likewise, no resonance of the spectrum is narrowed when ["Nlcyanide is used. Thus it appears that the unique EPR spectrum of the enzyme-PCA-cyanide complex results from chelation of the active site Fe3+ by PCA and simultaneous binding of cyanide to the enzyme but not directly to the iron.

On standing anaerobically at room temperature, the sam- ples used to evaluate the coordination of PCA and cyanide slowly convert to the low spin, final complex. The EPR spectrum of this complex is qualitatively identical to that of the complex formed in the absence of PCA. In contrast to the high spin enzyme-cyanide-PCA complex, hyperfine broaden- ing from [I3C]cyanide is observed in the low spin EPR reso- nances (Fig. 6C). No broadening from either 3-I70H- or 4- "OH-labeled PCA is observed (not shown). Likewise, no broadening is observed from 170-labeled PCA added to the low spin enzyme-cyanide complex formed without PCA pres- ent. Thus PCA is apparently excluded from the iron in the

Ligand Binding by Protocatechuate 3,4-Dioxygenme 8795

H FIG. 6. EPR spectra of the enzyme-cyanide-PCA complex

formed with ["OIPCA or [13C]cyanide. Enzyme (79 p M ) was anaerobically mixed with 100 mM KCN in 200 mM MOPS buffer, pH 7.0, and 20 mM PCA was added. The sample was frozen immediately to trap the intermediate enzyme-cyanide-PCA complex. A , complexes formed with unenriched (-----), 3-l'OH-enriched (long dashes), or 4- "OH-enriched (-) PCA. Note that the latter two species exhibit nearly superimposed spectra. B, complexes formed with unenriched (-----) or 13C-enriched (-) cyanide. C, sample from E was incubated for 74 h to allow formation of the final, low spin complex. Only a portion of the enzyme was converted during this period due to the relatively high concentration of PCA present (see inset spectrum). Spectra were measured under the conditions described in Fig. 1. The spectra in A are normalized to the same intensity. (4-I70H-enriched complex spectrum X 1.18 3-"OH-enrichedcomplex spectrum X 1.23.) The line shapes of the complete spectra are shown in the insets (see Fig. 5).

low spin cyanide complex. It is not known whether it is excluded from the active site.5

Steady State Kinetic Studies of Cyanide Complex Forma- tion-As shown in Fig. 7, cyanide is an inhibitor of proto- catechuate 3,4-dioxygenase. Since the steady state turnover occurs on a much faster time scale than the formation of the low spin, final enzyme-cyanide complex described above, it is likely that either the initial EPR detectable complex or one that causes no perturbation in the EPR spectrum is respon- sible for the inhibition. If the cyanide were functioning as a unimolecular competitive or noncompetitive inhibitor for PCA, one would expect the slope replots of the data shown in Fig. 7 to be linear. However, as shown in Fig. 8, this is not the case; the slope replot is concave upward. Such replots are

Occasionally reaction with cyanide in the absence of PCA yields two low spin complexes exhibiting similar EPR spectra. Addition of PCA to the sample causes the EPR spectra to coalesce into one species with the same g-values as usually observed. This leads us to believe that PCA does bind to the low spin cyanide complex of the enzyme but not directly to the iron.

0 1 -2.5 0.0 2.5 5.0 7.5 10.0 12.5

l / [ P C A ] ( r n M - l ) FIG. 7. Steady state kinetics of PCA turnover in the pres-

ence of cyanide. The initial velocity of PCA oxygenation by enzyme (0.1 PM sites) was measured using an oxygen electrode. NaCN was present in the following concentrations: 0 (e), 100 mM (A), 200 mM (W), 300 mM (V), 400 mM (e). Conditions: buffer, 100 mM sodium phosphate, pH 7.0; temperature, 31 "C; 0.250 mM 0,. The total concentration of SO:- in the solution was maintained at 300 mM by adding a combination of H,SO, (to maintain the pH) and Na,SO,.

100 lZ5 r"7l h

v v) 75

0) a 0

50

25

I 0.0 0.1 0.2 0.3 0.4 0.5

[NaCNI (MI FIG. 8. Slope replot of the kinetics of PCA turnover in the

presence of cyanide. The slopes of the curves shown in Fig. 7 are plotted as a function of total NaCN concentration. The solid line is a fit of the data to a parabolic function.

observed for the case in which two or more inhibitors act at different sites to competitively inhibit substrate binding, and the concentrations of the inhibitors are varied simultaneously in a constant ratio.

Ki I,E === I + E + PCA 6 E.PCA &E + product 0

+ I I

+

I a E I b e I + EI, K2

This situation would, for example, obtain if the inhibitor for both sites illustrated above were CN-. Application of the rapid equilibrium assumption yields a reciprocal initial veloc-

8796 Ligand Binding by Protocatechuate 3,4-Dioxygenase

ity equation of the following form (24):

where Ki and aK, are the two dissociation constants for the binding of the generalized inhibitor ( I ) . The occurrence of the 1' term in the numerator of the slope term of Equation 1 gives rise to the nonlinear slope replot. If the factor a is not large (or, in the case of two different inhibitors, if IJK, = Ib/aKi) the nonlinearity will be pronounced. If the number of com- petitive inhibitor sites is, in fact, two, then a parabolic slope replot is expected. The solid line in Fig. 8 is a parabolic fit to the data and shows that the assumption of two competitive inhibitors with similar [IJ/K, values is reasonable. Similar parabolic curves (not shown) are observed for variation of total cyanide concentration at pH values between 7 and 9, in MOPS and phosphate buffers, and in solutions containing a variety of added salts up to 500 mM.

Equation 1 predicts that the inhibitor will have no intercept effect on the double reciprocal plot. However, this is clearly not the case for the data shown in Fig. 7 (and Fig. 9, see below). The observed intercept effect suggests that an en- zyme-cyanide-PCA complex is possible. Moreover, the exist- ence of such a complex is consistent with the EPR results presented above. Thus, the intercept term of Equation 1 must be modified

Ki Z,E Z + E + PCA E. PCA- E + product

0 2

+ Z

+ + Z Z

&11 1 1 Z,,EZL,= + E I b + PCA"-EZb.PCA

Ki

The data presented above does not indicate whether the actual inhibitor is HCN or CN-. This can be investigated by

20

n 15 In I 3

> X

*I 10

V

5- " 5

0 -2.5 0.0 2.5 5.0 7.5 10.0 12.5

1/[PCA] (mM-l) FIG. 9. Steady state kinetics of PCA turnover in the pres-

ence of cyanide as a function of pH. Initial velocity of PCA oxygenation was measured as described in Fig. 7 at a constant NaCN concentration of 100 mM. The pH values of the 0.1 M sodium phosphate buffer were: 7.00 (0), 7.25 (A), 7.50 (U), 7.75 (V), 8.00 (a), 8.25 (A), 8.50 (M), and 8.75 (V).

measuring kinetic data a t a variety of pH values (Fig. 9). The assumption of a nominal pK, value of 9.31 for protonation of CN-, allows a slope replot of the data measured at different pH values to be made based on the calculated concentration of CN- (Fig. 10). If CN- were the inhibitor for both sites, a parabolic curve like that observed for variation of total cya- nide at a constant pH (Fig. 8) would be expected. Instead, the curve is approximately linear. This shows that, while CN- is an inhibitor of the enzyme at one site, it is not the inhibitor at the second site. If it is assumed that HCN is the inhibitor at the second site, Equation 2 must be rewritten in terms of two different inhibitors; for example, the [I]' term becomes [CN-][HCN]/aK,2. In the pH range investigated (pH = 7.0 to 8.75), the CN- concentration increases 43-fold while the HCN concentration decreases only 0.21-fold. Thus, to a first approximation, [HCN]/Ki can be considered a constant rela- tive to [CN-]/aK, in the slope term of Equation 2. Conse- quently, the predicted replot of slope uersus [CN-] would be linear as observed. At a constant pH, however, [CN-] and [HCN] would vary in a constant ratio as the total cyanide concentration changed. This would account for the observed parabolic slope replot seen in Fig. 8. The observed increase in the intercept of the double reciprocal plot with increase in [CN-] (Fig. 10) suggests that CN- is the form that binds simultaneously with PCA. The concentration of HCN de- creases with increasing pH, thus, according to Equation 2, the intercept would be expected to decrease if HCN functioned as the noncompetitive inhibitor. The apparent competitive inhibition of HCN implies only that HCN is the form which binds. Deprotonation to CN- on the enzyme surface may occur.

Approximate K, values for cyanide binding can be obtained by nonlinear regression fitting of the slope replot data shown in Fig. 10 using Equation 2 modified as described above and K,,,/V,,,, K,, and aKi as free parameters. Values of K,,,/V,,,., = 5.8 s, aK, (for CN-) = 1.7 mM, and K, (for HCN) = 90 mM allow a good theoretical fit to the data including the small observed deviation from linearity (solid line, Fig. 10). The intercept term of Equation 2 allows the Ki of CN- binding to

200 , , 20

h 150

I V

h

V VI 100

al a 0 rn 4

50

0 5 10 15 20 25

FIG. 10. Slope and intercept replots of the kinetics of PCA turnover in the presence of cyanide as a function of pH. The slopes (0) and intercepts (0) of the curves shown in Fig. 9 are plotted as a function of the calculated CN- Concentration. The solid line shown through the slope data was calculated using the best fit parameters determined using nonlinear regression analysis with Equation 2 (see text). The solid line shown through the intercept data is the linear least squares fit to the data.

0.9

h

E d 0 0.7 0 u3 v

0 0.5

n Lt 0 v)

2 0.3

0.1

Ligand Binding by Protocatechuate 3,4-Dioxygenase 8797

0 1000 2000

0 1000 2000 3000

Time (s)

FIG. 11. Kinetics of the formation of the final enzyme-cya- nide complex. Enzyme (60.5 PM) was mixed anaerobically in a closed vessel with 278 mM NaCN in the absence of PCA ( A ) or in the presence of 1.5 mM PCA ( B ) . The time courses of the reactions monitored at 500 nm are shown. The linearity of the inset plots shows that the reactions are pseudo-first order. Conditions: buffer, 500 mM MOPS, pH 7.0, plus 100 mM NazS04; Temperature = 31 "c.

the enzyme in the presence of PCA to be estimated as -20 mM from the ratio of the intercept and slope of the intercept replot shown in Fig. 10.

The activity of the enzyme is sensitive to both pH and ionic strength (5). Salts such as Na,SO, activate the enzyme while other salts such as NaCl are inhibitors. The pH and salt effects are minimized for the data shown in Figs. 9 and 10 because the specific activity maximizes in a broad plateau in the pH range between 7 and 9, and the salt concentration is held approximately constant. However, the effect of large changes in the salt concentration on the data shown in Figs. 7 and 8 is potentially substantial.6 Consequently, the kinetic parameters were derived from the data shown in Fig. 10.

Transient Kinetic Studies of Formation of the Final Cyanide Complex-As shown in Fig. 11, formation of the low spin, final cyanide complex with protocatechuate 3,4-dioxygenase is a well-behaved pseudo-first order process a t cyanide con- centrations high relative to that of the enzyme. In the presence of PCA, the reaction is slower as shown in Figs. 11 and 12, but remains pseudo-first order. The observed dependence on PCA is consistent with the scheme shown below in which formation of the initial cyanide complex is rapid, PCA binding is reversible and prevents formation of the final cyanide complex, and the formation of the final cyanide complex is effectively irreversible (as observed).

E + nZ F== Z,E + Z - (Z,)EZ (final complex) kz '

+PCA k-31 Ik:,

IE . PCA

As discussed above the formation of I,E is complex and

3.0

h m 0 4

x 2.0 4

'v)

Ao 1.0

v

n v)

0.0 1 0.0 1.0 2.0 3.0 4.0 5.0

W A I (mM) FIG. 12. Dependence on PCA concentration of the observed

rate of final enzyme-cyanide complex formation. Apparent first order rate constants determined using exponential nonlinear regres- sion fits to data like that shown in Fig. 11 is plotted uersuS unbound PCA concentration. The solid l ine was calculated from best fit param- eters determined using nonlinear regression analysis with Equation 3. A linear transform is shown in the inset. The rate constant for the reaction in 500 mM sodium phosphate buffer, pH 7.0 (0) is shown for comparison. The unbound concentration of PCA was estimated from the known active site concentration and the K d values for PCA in the presence of cyanide estimated from the data shown in Fig. 4 (bottom).

involves at least two cyanides. It is not known whether the cyanide that binds during the formation of the intermediate is retained in the final complex.

If the initial binding of cyanide is assumed to be saturated and the complex with PCA is assumed to be in equilibrium during the formation of the final cyanide complex, the follow- ing relationship is approximately true (25, 26).

where Kd3 (= k3'/k-3) is the dissociation constant of PCA for the intermediate enzyme cyanide complex and k,' is a pseudo- first-order rate constant that includes the cyanide concentra- tion. The solid line in Fig. 12 is the theoretical curve calculated by fitting the data with Equation 3. The values of the con- stants estimated from the data are: Kd3 = 820 WM and k,' = 0.003 s-l at 31 "C and 278 mM NaCN, pH 7.0.

If the scheme described by Equation 3 is correct, the ob- served rate constant k2' will exhibit a linear dependence on cyanide concentration. Such a dependence was not observed (Fig. 13). However, the square root of the observed rate constant does increase linearly with cyanide concentration (Fig. 13). This suggests that the binding of cyanide to form the final complex is second order in cyanide. Thus the for- mation of the final cyanide complex appears to involve the binding of two cyanide molecules in addition to any already bound to the enzyme.

Nonlinear regression fitting of the Fig. 8 data gives exactly the same values for the kinetic paramenters that relate specifically to cyanide inhibition if one of the following assumptions is made: 1) there is a rather nonselective inhibitor binding site for salt ions, requiring that Equation 2 is rewritten as a third order equation; or 2 ) K,,,/V,., is not a constant but rather increases linearly with the salt composition of the reaction mixture. We favor the latter assumption because a third order equation is not required to obtain an excellent fit of the Fig. 10 data.

E + nZ F= Z,,E + 21 - (I,) E1 (final complex)

ZE . PCA

Binding of 14C-Labeled Cyanide-The stability of the final enzyme-cyanide complex allowed direct quantitation of tightly bound cyanide. 14C-Labeled cyanide was reacted with

Ligand Binding by Protocatechuate 3,4-Dioxygenase

7.0 - 0 6.0 I i v

6 7 5.0

0 .+ 4.0 x

I 3.0 .-I

v) v

$ 2.0

4: 0

1.0

0.0

0.10

0.08 2 I 0 V

0.06 &in,

L ‘ v )

0.04

0.02

0.00 0.00 0.20 0.40 0.60

[NaCNI (M) FIG. 13. Dependence on cyanide concentration of the ob-

served rate of final enzyme-cyanide complex formation. Ap- parent first order rate constants were determined from data like that shown in Fig. 11. The apparent rate constants (0) and the square root of the constants (0) are plotted uersus cyanide Concentration. No PCA was present. Conditions and enzyme concentration were as described in Fig. 11.

the enzyme to form the final complex. Excess cyanide, as well as any rapidly exchangeable cyanide, was removed by gel filtration and the bound cyanide concentration was deter- mined from the known specific activity of the cyanide (0.94 Ci/mol). The iron content of the samples was independently determined by inductively coupled plasma emission spectros- copy. Five samples showed 1.8 k 0.4 tightly bound cyanides/ iron.

DISCUSSION

We have used hyperfine interactions between the electronic spin of protocatechuate 3,4-dioxygenase and the nuclear spin of 170-, 13C- or I5N-labeled ligands to investigate substrate and small molecule coordination. Such measurements provide unequivocal evidence for binding because through space in- teractions are not detectable (27) (for a discussion see Ref. 14). The results of our previous studies of protocatechuate 3,4-dioxygenase have been interpreted in the context of a mechanistic cycle which begins with formation of a PCA-Fe3+ active site chelate and proceeds through a peroxy-intermedi- ate in which PCA is ketonized and coordinated through only the 4-OH group. The iron ligand position vacated by the 3- OH group of PCA is proposed to be occupied by the distal oxygen of the organic peroxide (14). Such an organization of the active intermediate would require a substantial rotation of the substrate in the active site7 and, perhaps, the presence of a binding site on the protein for the ketonized 3-OH group of the PCA. The data described here are consistent with this model in three important respects. First, both PCA and the alternate substrate HPCA appear to bind to the iron as chelates in some complexes. Second, no more than two ligands appear to coordinate to the iron in any complex suggesting that there are only two ligand sites accessible to exogenous ligands. Thus, at least one site is shared by PCA and other ligands including, perhaps, the organic peroxide. Third, a

’ Rotation of the substrate is considered relative to the iron coor- dination. Changes in the geometry of the coordination could increase or decrease the postulated physical movement of the substrate relative to the overall structure of the protein.

cyanide binds at a protein site away from the iron to markedly perturb the substrate-iron interaction. If this site is near the iron, it could serve as a binding site for the postulated keton- ized 3-OH group, another substrate functional group, or oxy- gen during turnover.

Substrate Binding-We have shown here that the slow substrate, HPCA, can coordinate to the iron through both of its hydroxyl functions. I t is conceivable that the hyperfine broadening observed in the EPR spectrum derives from ap- proximately equal populations of two binding orientations that result in both the 3- and 4-OH groups occupying the same binding site on the iron. However, since the substrate is asymmetric, it is unlikely that two substrate orientations would result in the same EPR spectrum. Because the broad- ening is observed in the same sharp resonances regardless of the location of the I7O label, it is quite likely that HPCA can form a chelate complex with the iron in a manner consistent with the mechanistic model. The overall similarity of the complex EPR spectra elicited by HPCA and PCA binding to protocatechuate 3,4-dioxygenase suggests that they bind in a similar fashion. In contrast, despite their close structural similarity to PCA, inhibitors elicit very different EPR spectra, showing that the spectrum is very sensitive to binding config- uration (14). The dominant rhombic species (g = 9.68, 4.28) that gives rise to the broadened EPR resonances in the spectrum of the HPCA-enzyme complex is also present in the PCA complex, albeit in lower concentration. Thus it is rea- sonable to speculate that PCA also can form a chelate com- plex. Although the inherently large line width of the more axial HPCA-enzyme species (g = 6.5, 5.5) makes the obser- vation of broadening difficult, the data suggest that this species is monodentate, binding through the 4-OH group. In the case of PCA binding, a larger proportion of the EPR detectable species gives this type of spectrum. Thus a larger fraction may be monodentate. We have attempted to detect broadening in the complex of protocatechuate 3,4-dioxygenase with I70-labeled PCA without success. However, in this case, no resonance is sufficiently narrow and resolved from other resonances to rule out the possibility that broadening occurs but is undetected. Hyperfine broadening from labeled PCA is readily observable in the enzyme-cyanide-PCA complex dis- cussed below.

Inhibitor Binding-The observed binding of the single OH group of 4-HBA and 4-HPA to the iron is consistent with the mechanistic model and with resonance Raman measurements of the former complex in solution (10). The hydroxyl group of 3-HBA apparently does not coordinate directly to the iron. This result is consistent with resonance Raman data that failed to show enhancement of ring mode vibrations for this inhibitor (10). Previous studies of water binding by the active site iron showed that 3-HBA, but not 4-HBA, displaced water from the active site iron on binding (14). The current results suggest that 3-HBA displaces the water by steric effects rather than direct binding to the iron. 3-HPA apparently binds differently than 3-HBA since both the hydroxyl group of 3- HPA and water coordinate to the iron. It is possible that the 3-OH group is brought closer to the iron in this complex due to the added methylene group of the carboxyl side chain and that a site for water binding on the iron is made available by this reorientation. The large increase in the Kd for the binding of both monohydroxyphenylacetate inhibitors suggests that the putative reorientation is not energetically favorable rela- tive to the binding orientation of the monohydroxybenzoates.

Cyanide Binding-When considered together, the kinetic and spectroscopic measurements reported here clearly indi- cate that cyanide has several functionally and physically

Ligand Binding by Protocatechuate 3,4-Dioxygenase 8799

distinct binding sites on protocatechuate 3,4-dioxygenase. These sites appear to either directly or indirectly modulate substrate binding to the enzyme and can thus be used to probe this interaction. Three aspects of the cyanide binding reaction suggest that two of the binding sites it can occupy are coin- cident with the substrate binding sites on the iron. 1) The binding reaction is biphasic and each phase is inhibited by PCA. 2) The intermediate enzyme-cyanide complex differs from the enzyme-cyanide-PCA complex in that cyanide is apparently displaced from the iron in the latter. PCA is directly coordinated to the iron in this complex. 3) The final enzyme-cyanide-PCA complex probably has two cyanides bound to the iron and PCA is apparently displaced. The evidence that there are two cyanide ligands in the final complex is also based on several observations. 1) The kinetics for cyanide binding in this step are second order in cyanide. 2) The magnitude of the hyperfine broadening from ["C] cyanide is much larger in the final complex (1.2 mT) than in the intermediate (0.7 mT) and is the largest we have observed from this isotope, consistent with an additional cyanide li- gand. 3) The occurrence of low spin, non-heme iron is rare in biological compounds and is associated with the binding of two or more cyanides in non-porphyrin model complexes (28). 4) Two strongly bound radiolabeled cyanides are retained after gel filtration of the final enzyme complex. Nevertheless, the data do not completely exclude the possibility that cyanide binds in only one site on the iron, and the observed spin conversion and enhanced hyperfine coupling from [''Clcya- nide result from a major conformational change in the protein. Such a change might allow more efficient coupling of the hyperfine field with the electronic spin system.

A third cyanide binding site located off of the iron, yet in a position to perturb substrate binding, is suggested by the observation of hyperfine broadening from 170-labeled PCA but not from [13C]cyanide in the ternary complex with en- zyme. The presence of such a site in addition to the rapidly exchanging cyanide binding site on the iron is strongly sup- ported by the linear intercept and parabolic slope dependence on cyanide apparent in the steady state kinetic inhibition analysis. Moreover, the unique EPR spectrum of the enzyme- cyanide-PCA complex considered together with its typical, substrate complex-like, optical spectrum are consistent with this model. The optical spectrum of the enzyme arises from charge transfer interactions between the iron and deproton- ated phenolic hydroxyl groups of both protein tyrosinate and substrate ligands. These interactions would remain essentially unchanged as long as the substrate is coordinated and the iron ligands remain unchanged. On the other hand, the EPR spectrum is very sensitive to both the physical and electronic symmetry of the ligand field. Thus small perturbations in the substrate binding geometry can cause large changes in the EPR resonance positions. Such a change could occur due to the binding of cyanide near the iron coordination sphere.

The EPR spectrum of the enzyme-PCA-cyanide complex has a narrow line width suggesting that the species is rather homogeneous. This is in sharp contrast with the substrate complex formed in the absence of cyanide which is extremely heterogeneous. Thus cyanide appears to bind at a site that prevents substrate from assuming the wide range of orienta- tions which characterize the cyanide free complex.

A model for postulated binding sites of cyanide in the active site of protocatechuate 3,4-dioxygenase and the interaction of cyanide with substrate is illustrated in Scheme 3. The low rate of conversion to the final complex is consistent with a major change in the iron ligation. There are several possibil- ities for the ways in which such a change might occur. For

SCHEME 3

example, an amino acid R-group may be displaced or the iron may change from 5 to 6 coordinate in order to accommodate the putative second cyanide that causes the low spin conver- sion. Since PCA appears to compete for the same iron ligand sites as cyanide, a similar structural rearrangement of the active site may occur during substrate binding.

Mechanistic Implications-Recent studies by Que and co- workers (29) have shown that intradiol oxygenolytic cleavage of 3,5-di-tert-butyl catechol is catalyzed by Fe3+ model com- plexes in which the catechol initially forms a chelate complex similar to that apparently formed by the enzyme with sub- strates. One important aspect of catalytically active model compounds appears to be that one Fe3+-0 bond to the catechol is longer than the other, allowing it to be preferentially broken as the catechol passes through a ketonized intermediate dur- ing the reaction (30). The results reported here indicate that the bond of the 3-OH group of PCA to the active site iron is similarly weakened relative to that of the 4-OH. In the case of the enzyme, the weakening could result from either the geometry of the iron coordination sphere which makes one bond longer, or constraints placed on the substrate by the structure of the active site pocket.

The results presented here suggest that PCA and cyanide (and water (14)) are mutually exclusive iron ligands. Conse- quently, no separate site reserved for small, oxygen-like mol- ecules appears to exist in the iron coordination. The opposite conclusion was reached from related studies of the active site structure of extradiol catecholic dioxygenases (18). These studies showed that there is a special ligand position on the active site Fez+ which is accessible to nitric oxide, an oxygen analog. PCA appears to be able to form a chelate complex with the iron, but it does not displace the nitric oxide; in fact, nitric oxide binding is strengthened (31). Thus there appear to be three iron ligand sites accessible to exogenous ligands, one of which is reserved for small molecules. We have postu- lated that extradiol dioxygenases activate oxygen by directly coordinating it to the iron in this special site (18). In the mechanism that we have proposed for the intradiol enzymes (14,32), there is no need for such a site because the substrate is activated through the formation of a complex with the Fe3+ for direct attack by oxygen.

The apparent presence of a binding site for cyanide off of the iron could have mechanistic significance, especially if it is in the active site pocket. This site might be coincident with the PCA carboxylate binding site. However, we believe that this is unlikely due to the greatly decreased heterogeneity of the enzyme-PCA-cyanide complex relative to the analogous complex without cyanide. The opposite result would be ex-

8800 Ligand Binding by Protocatechuate 3,4-Dioxygenase

SCHEME 4

pected if one of the primary binding determinants for PCA were blocked. The increased homogeneity does suggest, how- ever, that the PCA molecule is constrained in some manner in the active site. For example, the binding of cyanide could fill residual space in the active site, limiting substrate move- ment at a single binding locus. Such a cyanide binding site might serve to bind oxygen prior to substrate attack during turnover. Alternatively, substrate may have two or more significantly different binding loci, one of which is blocked by cyanide. For example, substrate binding, like cyanide binding, may be a multistep process. The cyanide binding site might serve to bind one of the OH groups of PCA in an intermediate complex. This site could then also stabilize the “rotated” orientation of the substrate required by the proposed mecha- nism in order to allow the distal oxygen of the organic peroxide intermediate to coordinate to the iron. One model which illustrates the latter proposal is shown in Scheme 4.

More information about the rapidly exchanging cyanide binding sites, including the site off the iron can be obtained through transient kinetic and spectral studies of the initial cyanide binding reaction that are currently in progress.

Acknowledgments-We gratefully acknowledge the assistance of J. Dege and W. Froland in the preparation of the enzymes and labeled substrates. We also thank M. Harpel for insightful reading of the manuscript.

REFERENCES

1. Stanier, R. Y., and Ingram, J. L. (1954) J. Biol. Chem. 210,799- 808

2. Chapman, P. J. (1972) Degradation of Synthetic Organic Molecules in the Biosphere, pp. 17-55, National Academy of Sciences, Washington, D.C.

3. Dagley, S. (1984) Deu. Ind. Microbiol. 2 5 , 53-65 4. Fujisawa, H., and Hayaishi, 0. (1968) J. Biol. Chem. 243 , 2673-

5. Whittaker, J. W., Lipscomb, J. D., Kent, T. A., and Munck, E. 2681

(1984) J. Bid. Chem. 259,4466-4475 6. Bull, C., and Ballou, D. P. (1981) J . Bid. Chem. 256 , 12673-

12680 7. Durham, D. R., Stirling, L. A., Ornston, L. N., and Perry, J. J.

(1980) Biochemistry 19, 149-155 8. Que, L., Jr., Lipscomb, J. D., Zimmermann, R., Munck, E., Orme-

Johnson, N. R., and Orme-Johnson, W. H. (1976) Biochim. Biophys. Acta 452 , 320-334

9. Que, L., Jr. (1983) Adu. Znorg. Biochem. 5 , 167-199 10. Que, L., Jr., and Epstein, R. M. (1981) Biochemistry 2 0 , 2545-

2549 11. Felton, R. H., Cheuna, L. D., Phillips, R. S., and May, S. W.

(1978) Biochem. Biophys. Res. Commun. 85,844-850 12. Keyes, W. E., Loehr, T. M., and Taylor, M. L. (1978) Biochem.

Biophys. Res. Commun. 8 3 , 941-945 13. Felton, R. H., Barrow, W. L., May, S. W., Sowell, A. L., Goel, S.,

Bunker, G., and Stern, E. A. (1982) J. Am. Chem. SOC. 104,

14. Whittaker, J. W., and Lipscomb, J. D. (1984) J. Bid. Chem. 259 ,

15. Whittaker, J. W., and Lipscomb, J. D. (1984) J. Bid. Chem. 259 ,

16. Lauffer, R. B., and Que, L., Jr. (1982) J . Am. Chem. SOC. 104,

17. Arciero, D. M., Lipscomb, J. D., Huynh, B. H., Kent, T. A., and

18. Arciero, D. M., and Lipscomb, J. D. (1986) J . Biol. Chem. 261 ,

19. Freifelder, M. (1971) Practical Catalytic Hydrogenation, pp. 168- 173, John Wiley & Sons, New York

20. Blumberg, W. E. (1967) in Magnetit Resonance i n Biological Systems (Ehrenberg, A,, Malmstrom, B. G., and Vannghrd, T., eds) pp. 119-133, Pergammon Press, New York

21. Blumberg, W. E., and Peisach, J. (1973) Ann. N. Y. Acad. Sci.

22. Aasa, R., and Viinnghrd, T. (1975) J. Magn. Reson. 19, 308-315 23. Bevington, P. R. (1969) Datu Reduction and Error Analysis for

the Physical Sciences, pp. 237-239, McGraw-Hill, New York 24. Segel, I. H. (1975) Enzyme Kinetics, pp. 176-178, John Wiley &

Sons, New York 25. Hiromi, K. (1979) Kinetics of Fast Enzyme Reactions, pp. 206-

212, Halsted Press, New York 26. Lancet, D., and Pecht, I. (1976) Proc. Natl. Acad. Sci. U. S. A.

73,3549-3553 27. Simanek, E., and Sroubek, Z. (1972) in Electron Paramagnetic

Resonance (Geschwind, S. ed) pp. 535-574 Plenum Press, New York

28. Cotton, F. A., and Wilkhson, G . (1980) Advanced Inorganic Chemistry, p. 761,4th Ed, John Wiley & Sons, New York

29. Que, L., Jr., Kolanczyk, R. C., and White, L. S. (1987) J. Am. Chem. SOC. 109,5373-5380

30. White, L. S., Nilsson, L. H., Pignolet, L. H., and Que, L., Jr. (1984) J. Am. Chem. SOC. 106,8312-8313

31. Arciero, D. M., Orville, A, M., and Lipscomb, J. D. (1985) J. Bid. Chem. 260,14035-14044

32. Que, L., Jr., Lipscomb, J. D., Munck, E., and Wood, J. M. (1977) Biochim. Biophys. Acta 485,60-74

33. Ohlendorf, D. H., Lipscomb, J. D., and Weber, P. C. (1988) Nature 336,403-405

6132-6134

4487-4495

4476-4486

7324-7325

Munck, E. (1983) J. Biol. Chem. 258 , 14981-14991

2170-2178

222,539-560

Ligand Binding by Protocatechuate 3,4-Dioxygenase 8801

SUPPLEMENTARY MATERIAL TO

B i n d i n g o f i s o t o p i c a l l y L a k l e d substrates. i n h i b i t o r s and CYanidQ by Protocatechuate 3.4 oioxygenase

~ l l e n M. O r v i l l e and John 0. Lipscomb

MATERIALS AN0 METHODS

Source of the enzymes- PrOtoCateChUste 3.4 dioxygenare and 4-hydroxyphenylacetate 3- hydroxylase were ~ m l a t e d from 8revibaCterrm furcum (ATCC 15991). Protocatechuate 3.4 diaxygenare was induced by growth on 4-HBA and p u r i f i e d a s previously descr ibed (51 Cu l tu r i ng o f 8. f u s c m under the r a m growth condi t ion wi th 4-HPA (2 9/11 IS the so le carbon source resu l ted r n t h e i n d u c t i o n o f I ~ h y d r 0 x y p h e n y l ~ c e t l t e 3 - h y d r o x y l a s e and homopmtocatechuate 2.3 dioxygenare I S wel l as protocatechuate 3.4 dioxygenase.

Enzyme d31ay1 and i ron con ten t - O iaxygenare ac t i v i t y was assayed by measuring Substrate dependent oxygen consumption ullng a Clark type oxygen electrode which had been c a l i b r a t e d by the rap ld d iaxygenat ian o f 0.1 irml o f PCA catalyzed by 0.2 Wml protocatechuate 3 4-diaxygenare (17). A typical assay mixture contained 1 mM PCA i n 50 nll MOPS b u f f e r pH'7.0 at 23 'C. Manooxygenare ac t i v i t y o f t he 4 -hyd roxypheny lace ta te 3 - hydroxylase was ar rayed e i t he r by m o n i t o r i n g t h e l o s s i n O p t i c a l absorbance at 340 nm due to the disappearance o f NADPH or by mon i to r ing the oxygen consumption a s above. The

consisted o f I nll 4-HPA and 0.1 nll NADPH 1 0 50 nll MOPS b u f f e r pH 7.0 a t 23 O C . The t y p i c a l assay mix tu re used f a r measurement of oxygen consumption by the hydroxylase

concentration O f re3+ ( a c t i v e r i t e s ) i n t h e p r o t o c a t e c h u a t e 3.4 dioxygenase #a$ c11 u la ted f rom the Opt ica l spectwm us ing a mola r ex t i nc t i on coe f f i c i en t O f 2900 M ~ I c K f at 434 nm ( 5 ) . H o l o e n z y ~ has a r u b u n i t s t r u c t w e O f ( . B F ~ ) ~ and the 5 a c t i v e rlter are apparently independent.

Mounds Labora tar le r , KOR l nc . and ICON Inc . and d i s t i l l e d once before use. l vO- labe led chemicals- Samples O f water enliched to minimum o f SO atom X "0 were obtained from

PCA, 3-H8A. and 4-HBA were prepared as prev iaur ly descr ibed (18). Cyanide enriched to 99 atom X i n ,,C or l s N was obtained from ICON Inc. All other chemicals were reagent grade. Water was de ion t red and then g l a r r d l r t i l l e d .

(50 rm. 0.331 m o l ) was added t o 0.660 m l I W e n r i c h e d water and then 0.060 m1 cHCL

dropv ise add i t ion ( to ta l t ime appmximacely 30 min) o f c o l d 4 M NaNO, (25 mg NaNOI. 1.2 (0.744 m o l . 2.2 equ iva len ts ) was added. The d iazo sa l t was formed by the slow.

equ iva len t i n 0.091 ml ["O lua te r ) , wh i l e t he en t i re so lu t i on was maintained below a temperature of 5 'C. A Crysta l O f urea was added to F a c t w i t h t h e excess HONO. The d iazon ium sa l t was hydro l yzed i n a m i c w r e f l u x apparatus containing a m i x t u r e o f 0.060 ml cHCL and 0.660 m1 [ ' ' O l w t e r at 95 OC. The co ld d iazon ium sa l t was added i n 0.060 ml I l i q U O t l t h r o u g h t h e condenser; each IUCCePsive a l i q u o t was added a f t e r t h e e v o l u t i o n o f HI had slowed. The i n i t i a l h y d r o l y s i s s o l u t i o n was deep orange and became pa le orange I 5 to 30 mi" a f t e r t h e l a s t a d d i t i o n o f t h e s a l t . The react ion vessel vas then cooled to l i q u i d N2 temperatwe and Connected to d m i c r o d i s t i l l a t i o n a p p a r a t U I f o r l e c o v e r y o f the ["Oluater. f o l l o w i n g d i r t r l l a t i o n . t h e r e s i d u e was d i s s o l v e d i n 2 ml water wi th 1 equ iva len t O f NaOH added. decolorized. and f i l t e r e d i n t o a test tube which contained 3 equ iva len ts HC1. D ie thy l e ther was used to extract the product and. upon removal o f t he

of the O f the product were i d e n t i c a l t o t h o s e o f authentic 4-HPA. Mars spec t ra l Io lven t , an o f f - w h i t e powder was obtained. The Opt ical spectra and HPLC re ten t i on t ime

m a l v r i r revealed an enrichment o f 1'0 in the hvdraxvl which was i n c lose mreement w i t h

Synthesis of 4-'700-enriched 4-HPA- Decolorized and r e c r y s t a l l i z e d 4-NH,-phenylacetate

conducted using a a n a l y t i c a l reverse phase A l tex ODs column e q u i l i b r a t e d i n a water: HPLC repara t ion and p u r i f i c a t i o n Of 170-cnriched compounds- HPLC SeParatiOnl were

i sopropano l : ace t ic ac id m ix tuw (89:lO:l). S a w l e r were e lu ted i socra t ica1 lY w i th the same so lvent m ix tu re . frictions were Col lec ted and pooled af ter palsage through the de tec tor . The organic products were i s o l a t e d by evaporating the solvent. Organic products frm appvox imte ly 10 separations were pooled and provided adequate mater ia l to conduct the expwiments reparted here.

Anaerobic procedures and EPR sample prepara t ions- Enzyme sampler were made anaerobic by repeated cyc ler o f evacuat ion and f l ush ing w i th argon from which oxygen had been removed by passage over 1 BASF Inc . copper Catalyst at 150 'C. EPR r m p l e r prepared to test for hyperf ine broadening f rom [ I 'OIwater were made by adding "0-enriched or unenrlched water to l y o p h i l i z e d enzyme conta in ing WPS t o make I LOO lrll pH 7.0 buffer so lu t ion *hen rehydrated. The pH O f both unenriched water and [170]uater were adjusted to 7.0 immediately before addi t ion. Opt ical spectra of the matched r m p l e r were recorded t o u e n f y t h a t t h e metal center concentrat ions were the sane. D i t h i o n i t e and o ther

d i l u t i o n s o f t h e o r i g i n a l sampler. reagents w e ~ e added I S concentrates I " ""enriched-water and represented on ly s l igh t

EPR rpec t ro rcepy- EPR spectra were recorded at X-band on a Varian E-I09 spectrometer w i t h an Oxford Inrtrumentr ESR-IO l i q u i d h e l i u m c r y o s t a t . Temperature and 9-va lue c a l i b r a t l o n r were a$ prev iowly descr ibed (17) . Oata was recorded f a r i n t e g r a t i o n and w b t r a c t l o n procedures with a d i g i t a l computer in ter faced d i rect ly to the spect rometer .

EPR spect ra o f enzyme bound, h igh sp in f e r r i c i on complexer w i th i so top i ca l l y l abe led I l gandr i n t he me ta l coo rd ina t i on were analyzed using the spin Hamiltonian:

where 0 and E/O are ze lo f i e l d s p l i t t i n g p i r m M t e r r A i s t h e t r a n s f e r r e d h y p e r f i n e coupling tensor of the i so top ica l l y labe led l igand. 'anb the o ther paraneter r haw the i r u s u a l d e f i n i t i o n s . The hyper f ine term I ' A ' 5 descr ibes the in te rac t ion o f the sp in w i th the nuc lear sp in o f the l igand ( I - 5/$ f w "0. I - 1/2 f o r "L. I - I f o r ,*N, I = 1/2 f a r I 6 N ) . I n p r i n c i p l e , t h i s t e r m r e s u l t s i n s p l i t EPR relonmces. however, the

observed as a broadening (na r row ing i n t he case s f "N) o f the s igna l . The tern E/O 1 % s p l i t t i n g i s not resolved i n t h e s p e c t r a r e p o r t e d here, and the e f fec t o f there terms i s

a nea~ure O f the departure sf the e lectronic environment Of t h e i r o n from a x i a l r y m e t r y . A p e r f e c t l y a x i a l c e n t e r g i v e r and E/D va lue o f 0 wh i le a maximally rhombic center g iver a va lue o f 1/3 (20,21). The E/D value can be determined d i r e c t l y from the o - v a l u e r . Q m n t i t a t i o n o f EPR IDectra was Derformed usin4 the procedure of Aara and

oxygen e l e c t r o d e i n w h i c h s u f f i c i e n t s o l u t i o n was used to completely fill the react ion chamber. The chamber was capped t o prevent gar exchange with the herd space.

were pwpwed anaerob ica l l y and t i t r a t e d to pH 7.0 w i t h NaOH. Cyanide r a l u t l o n r were Temperature was maintained at 31 "C w i t h I c i r c u l a t i n g water bath. Substrate solut ions

prepared by adding LOO nll WPS or sodium phosphate b u f f e r t o s o l i d NaCN i n 1 rerum

des i red va lue w i th H,SO.. NalSO. was added t o b r i n g t h e t o t a l s u l f a t e c o n c e n t r a t i o n t o stoppered v i a l . The pH M I S moni tored wi th a 9 1 1 1 ~ e l e c t r o d e and was adjusted to the

300 nM. 1.5 ml of the cyanide containing solut ion was t r ans fe r red t o t he oxygen e lec t rode chamber f o r each reac t ion . A l iquot$ (1-25 "1) PCA so lu t ions were t rans fer red to the react ion vessel using gas t ight syr inges. React ions w e w s t a r t e d by a d d i t i o n o f

Na,SO.. The i n c l w i o n o f NaSO. i n t h e b u f f e r s i s known to be n e c e s ~ a r v to o~ercome an 2 o f concentrated enzyme e q u i l i b r a t e d ~n 100 nll MOPS b u f f e r pH 7.0 p lus 300 nll

K i n e t i c measurements- Steady s t a t e k i n e t i c s were measured i n d u p l i c a t e u s i n g an

i o i i c . r t r e n g t h dependence o f - w b s t r a t e b i n d i n g ( 5 1

spectrophotometer. Temperature *PI maintained at 31 OC w t h a c i r c u l a t i n g water bath. Translent k l n e t i c mearurementl were made using a Hewlett Packard 845111 diode away

Substrate and cyanide so lut ions were prepared anaerobical ly. Enzyme was p l a c e d i n an anaerobic Cuvet te and the appropriate a m a n t s o f PCA and cyanide added from gar t i g h t syringes. At the end o f the reac t ion , the PCA concentrat ion i n t h e Cuvette was determined by rap id oxygenat ion w i th added protocatechuate 3.4 dioxygenare. Oxygen uptake was measured p o t e n t i o m e t r i c a l l y a s described above. Data recorded by the

'Onstants were determined d i r e c t l y f rom the data wing I n o n l i n e a r r e g r e s s i o n f i t t i n g spectrophotometer YIP t r a n s f e l l e d t o I labsratO?y Cornputel fo? f u r t h e y a n a l y r i r . Rate

procedure (15,231.

RESULTS

TABLE I

"0-Ligand binding by protocatechuate 3.4 daoxygenare'

t h e i t a r t i n g enrichment of 1.0 i n t h e water

Ligand E/D 9-value 170-Ligand Ligation [170]water L i g a t i o n l i n e width/bb l i n e width/bb

(mT1 1.T)

was s i m i l a r t o t ha t p rev ious l y desc r ibed fo r the synthesis of [ 4 - h y d r 0 x y l - ~ ~ o ] P C A 118). Synthesis o f 3 - j 7 0 . and k-1700-enr i~hed HPCA- The synthesis o f [4-hydroxyl-"O]HPCA

k- [hydraxy l -~ 'O lHPA was conver ted enzymat ica l ly to [ 4 - h y d r o x y l - ~ 7 0 ] H P c A u t i l i z i n g 4-OH pheny lace ta te 3 -hydraxy la re . A ca ta ly t i c Concentmt ion o f NABPH was maintained by using I regenera t ing ry r tm o f g lucose-6-phosphate and glucose-(-phosphate dehydrogenase. The hydroxy la t ion reac t ion mix tu re con ta ined 56 rU 4-[hydroxyl-~70]HPA. 22 nll g luco le -6- phosphate. 1 . 8 NADPH, i n 2.8 ml O f IO0 1111 WPS pH 7.0 conta in ing 1 nll 2-morcaptoethanol and 10 d4 FAD. Af ter ad justed the pH t o 7.0 w i t h 6 N KOH. 50 u n i t s Of ca ta lase (Sigma) and 10 u n i t s Of glucole-6-pholphate dehydrogenase uele added. The hyd roxy la t i on reac t i on was i n i t i a t e d by the add i t i on o f 15 un i ts o f the 4 -hydroxy

enriched "0-enriched 0, and unenriched 4-HPA *ere used. The vessel was evacuated and The [P-hydroryl-~'O]HPtA was synthesized i n a r i m i l w manner except t h a t 50 atom X

f lushed several t imes wi th argon. and then one a t m s p h e r e o f "0, was added. The enzyme and reagent IOlUtions added to t h e f l a s k were made anaerobic. After completion of the

w i t h l i q u i d N,. An "0-enrichment o f 49% was m a w r e d by mass s p e c t r a l a n a l y s i s o f t h e reac t ion , the "0, was recoveled by condensing the gar i n a storage f l ask coo led to 77 K

decarboxylated fragment ion.

Synthesis of IJ70Jc.rboxyl,te-cnriched HPCA- HPCA l a b e l e d i n t h e c a r b o x y l g v w p w i t h "0 was synthesized ill prev ious ly repor ted fo r the syn thes is of [carboxylate-"I)lPCPIO]PCA

were sealed under vacuum and heated a t I10 'C ior 24 h. The enriched water was 1181. 50 "XJ o f HPCA. 550 *l O f [x70]water 51 S X enr i ched i n "0) and 50 "1 of cHCL

recavered by l y o p h i l i z a t i o n . Mars s p e c t r a l r n r l y s i l showed t h a t t h e exchange was complete and y i e l d e d t h e s t l t i l t l c l l d i s t r i b u t i o n o f s i n g l y % d d o u b l y l a b e l e d c a r b o x y l g r o u p ~ . NO ether exchange of hydroxyl oxygen was observed.

Nat ive Enzyme

None 0.33 9.67 - 1.4/0.2 YesC

Substrate Complexes

3~[OH~1701HPCA 0.33 4.28 1.75/0.38 Yes 1.9/0 Na

4~[OH~I7O1HPCA 0.33 4.28 1.7S/0.10 Yes 1.9/0 NO 0.02 5 . 5 1 Yes NO

[COOH~I7O1HPCA 0.33 4.28 1.75/0 No 1.9/0 NO NO

0.02 5.51 NO NO

0.02 5 .51 NO

I n h i b i t o r Complexes

4~[OH~I7OIHBA 0.32 4.28 1.6/0.1 yes l.6jO.3 yes 0.32 9.65 e 0.24 4.83 6.3/0.8 6.3/0 0.24 9.30 1.9/0 1.9/0.16

3-IOH-1701HBA 0.33 4.28 1.610 0.33 9.67 1 .5 /0 No ;:;;: 0.25 k.76

d d

4-[OH-170]HPA 0.339 9.67 1.4/0 NO 1.4/0.2 Yes 0.20 9.08 2.2/O.l5 yes 2.2/0 0.20 5.10 4.4/0.2 3.9/0

3-[OH-I7O1HPA 0.339 9.67 1.4/0 NO 1.k/O.2 Yes 0 .23 9.28 1.7/0.7 0.23 4.88 4.4/0.6 Yes : : i f : : Y e s

Enzyme was mixed w i th e i ther IO nll HPCA or 50 nll i n h i b i t o r a n a e r o b i c a l l y . E i t h e r t h e l i g a n d or water was en l i ched i n "0 for compwi lon w i th the unen l iched cont ro l . Llne width o f the reronance in the unenr iched cont ro l and t h e d i f f e r e n c e i n l i n e w i d t h VI. the same resonance i n t h e 1.0-enriched 1amo1e. From r e f . ( 1 4 ) Resonance i s too broad f o r unequivocal detection of broadening.

No broadening i s observed i n t h i s o r my assoc ia ted resonance although overlapping resonances prevented quant i t ia t ion.

e Broadening i s observed although overlapping resonances prevented quant i ta t ion.

9 Assigned a$ enzyme which has not bound i n h i b i t o r .