[l'o]water and nitric oxide binding by protocatechuate 4,b

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc.

Vol. 260, No. 26, Issue of November 14035-14044 1985 Printed in d.S.A.

[l‘O]Water and Nitric Oxide Binding by Protocatechuate 4,B-Dioxygenase and Catechol 2,3=Dioxygenase EVIDENCE FOR BINDING OF EXOGENOUS LIGANDS TO THE ACTIVE SITE Fez+ OF EXTRADIOL DIOXYGENASES*

(Received for publication, April 22,1985)

David M. Arcierog, Allen M. Orville, and John D. Lipscomb8 From the Department of Biochemistry, Medical School, University of Minnesota, Minneapolis, Minnesota 55455

Pseudomonas testosteroni protocatechuate 4,5- dioxygenase and Pseudomonas putida catechol 2,3- dioxygenase (metapyrocatechase) catalyze extradiol- type oxygenolytic cleavage of the aromatic ring of their substrates. The essential active site Fe2+ of each enzyme binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = %. Hyperfine broadening of the EPR resonances of the nitrosyl complexes by ‘?O-enriched H20 shows that water is bound directly to the Fez+ in the native enzymes, but is apparently displaced in substrate complexes. NO is not displaced by either substrates or inhibitors. The EPR spectra of several enzyme-inhibitor-NO complexes are different from those of enzyme-NO or enzyme-substrate-NO complexes and are found to be broadened by “0-enriched water. The data show that at least 2 and perhaps 3 sites in the Fe ligation can be occupied by exogenous ligands. Furthermore, it is likely that substrates and inhibitors displace water by binding either at or near to the Fe in the nitrosyl complex.

Nitric oxide binding is found to be substrate-dependent for each enzyme. Native catechol 2,3-dioxygenase exhibits KO values of 190 PM and 2.0 mM for NO binding in two types of independent sites. Only one type of site is observed in the catechol complex which exhibits a &for NO of 3.4 p ~ . One type of NO binding site is observed for both the native and substrate com- plexed protocatechuate 4,5-dioxygenase with KO values of 360 and 3 PM, respectively. The presence of a specific site in the Fe coordination for NO which is modified in the substrate complex, suggests that O2 binding by the extradiol dioxygenases may also occur at the Fe.

Dioxygenase enzymes which catalyze aromatic ring cleavage of catechol, PCA’ and related compounds are broadly classi-

* This work was supported by United States Public Health Service Grant GM24689 from the National Institute of General Medical Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

t Recipient of National Institutes of Health predoctoral trainee- ship in partial support of this work. Current address: Dept. of Micro- biology and Immunology, University of California, Berkeley, Berke- ley, CA 94708.

§ To whom all correspondence regarding this manuscript should be sent.

The abbreviations used are: PCA, protocatechuic acid (3,4-dihy- droxybenzoic acid); MOPS, 3-(N-morpholino)propanesulfonic acid; salen, N,N’-ethylenebis(salicy1adimine); 2-OH-INO, 2-hydroxyisonicotinic acid-N-oxide; 6-OH-NNO, 6-hydroxynicotinic acid-N- oxide.

~~~~~~~~

fied as intra- or extradiol according to the site of ring opening relative to the ortho-hydroxyl groups of the substrate (1).

R=COOH, H

Intradiol dioxygenases utilize a tightly bound, active site Fe3+ in a catalytic mechanism which apparently involves attack of O2 directly on the substrate (2-4). The structure and mechanism of these enzymes has been investigated through the use of many different spectroscopic techniques. Studies have revealed several of the Fe ligands (5-8) and have suggested the chemical nature of some of the reaction cycle intermediates (9, 10). In one such study, data from our laboratory has been used to show that two of the ligand sites of the active site Fe3+ of protocatechuate 3,4-&oxygenase iso- lated from Brevibacterium fuscum can be occupied by exogenous ligands (4). At least one of these ligands is water in the native enzyme as shown by hyperfine broadening of the EPR spectrum of a sample prepared in 170-enriched H,O.

In contrast to the intradiol enzyme studies, little progress has been made in the study of the mechanism of extradiol dioxygenases, such as protocatechuate 4,5-dioxygenase (11, 12) from Pseudomonas testosteroni and catechol 2,3-dioxygen- we (13-15) from Pseudomonas putida. This is due primarily to the fact that these enzymes contain an active site Fez+ which exhibits no useful optical or EPR spectra (12,14). Only Mossbauer spectroscopy has been successfully used in broadly characterizing the active site Fe (12, 16). As a result, the Fe Iigand structure and the steps of the reaction cycle are un- known.

Recently, it has been reported from our laboratory and others that the nitric oxide (NO) complexes of Fez+ mono- nuclear, nonheme Fe proteins can be effectively used to probe the Fez+ center by EPR spectroscopy (12,17,18). The nitrosyl complex of protocatechuate 4,5-dioxygenase (12, 17) as well as those of soybean lipoxygenase (19) and putidamonooxin (20) from P. putida have an electronic spin of S = 3/2 and exhibit EPR spectra with characteristic g values near g = 4

14035

14036 ("01 Water and NO Binding by Extradiol Dioxygenases

andg = 2. The intense S = 2/3 type EPR signal is also observed, albeit markedly altered, in ternary complexes of the dioxygenase enzymes with NO and substrates or inhhkors, suggesting that the latter molecules do not compete for the NO binding site on the iron (12).

In the current study, we have prepared the nitrosyl complexes of protocatechuate 4,5-dioxygenase and catechol 2,3- dioxygenase in 170-enriched water, and find that the EPR spectra are broadened showing that water is bound to the Fe simultaneously with NO. Ternary complexes with substrates and some inhibitors result in loss of the broadening suggesting that the water is displaced by these molecules. Other inhibitors, particularly those capable of only monodentate Fe ligation, are found to alter the EPR spectrum of the nitrosyl complex but do not displace the water. The affinity of each enzyme for NO is found to be markedly increased in the substrate complex, suggesting that the substrate and NO binding reactions are energetically coupled. This is the first direct demonstration of ligand binding of any type to the Fez+ of extradiol dioxygenases. The data strongly support binding of substrates and inhibitors at or near the iron. Furthermore, the data show that, like intradiol dioxygenases, the extradiol enzymes have two or more sites in the Fe coordination which are accessible to exogenous ligands and could be occupied by substrates and O2 during the catalytic cycle.

MATERIALS AND METHODS

Protocatechuate 4,5-dioxygenase was purified from P. testosteroni strain Pt-L5 grown on 4-hydroxybenzoate as described previously (12). Protocatechuate 4,5-dioxygenase enriched in 57Fe was prepared from cells cultured in media containing 57Fe as described previously (12). The buffer used for all experiments with protocatechuate 4,5- dioxygenase was 50 mM MOPS, pH 8.2, plus 10% glycerol. Catechol 2,3-dioxygenase was purified from P. put& (formerly Pseudomonas aruilla, ATCC 23973) maintained on mta-toluic acid and grown on benzoate as the sole carbon source. The buffer used in all cases, except where noted, for catechol 2,3-dioxygenase was 50 mM KP04, pH 7.5, plus 10% acetone. The purification was as previously described (15) except that after the DEAE chromatography step the enzyme was brought to 0.4 M in ammonium sulfate in buffer, and loaded onto a phenyl-Sepharose column (2 x 20 cm). After washing the column with buffer containing 0.2 M ammonium sulfate, the enzyme was eluted with a decreasing linear gradient of ammonium sulfate (0.2 to 0 M) in buffer. The enzyme was eluted at approximately 0.05 M ammonium sulfate. The enzyme collected from this procedure showed a single band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequent crystallization of this enzyme did not increase the specific activity which was found to be between 250 and 320 pmol min" mg-l in several preparations. The enzyme used to obtain the data presented here was not crystallized, however, the results were found to be unchanged in parallel experiments in which the crystallized enzyme was used. The enzyme was found to contain approximately 3 Fe2+ atoms/molecule. Catechol 2,3-dioxygenase enriched in 57Fe was prepared from bacteria which had been cultured in media containing 1 mg/l of 90% enriched 57Fe as the sole source of iron.

Samples of water enriched to 52.7 atom % 1 7 0 were obtained from Mound Laboratory. Water enriched to 99 atom % in "0 was obtained from KOR. The water was distilled once in a microdistillation appa- ratus before use. This was found to be necessary in order to remove an unidentified impurity which resulted in partial destruction of the metal centers. PCA was prepared by the method of Pearl (21). 4- Sulfonylcatechol was synthesized by the method of Ray and Dey (22). The rnetu-toluic acid, substrate analogs, and inhibitors were supplied by Aldrich or Sigma and were recrystallized before use. 2-Hydroxyiso- nicotinic acid-N-oxide and 6-hydroxynicotinic acid-N-oxide were synthesized by J. Whittaker using procedures described previously (10). 57Fe was supplied by New England Nuclear. Nitric oxide was supplied by Matheson Inc., East Rutherford, NJ. Enzyme grade ammonium sulfate was supplied by Schwarz/Mann. All other chemicals and growth media reagents were reagent grade and used without further purification. Water was deionized and then glass distilled.

EPR spectra were recorded on a Varian E-109 spectrometer equipped to make measurements near 4 K and record spectra digitally as previously described (12). Enzyme activities were determined using a Clark type oxygen electrode supplied by Yellow Springs Instrument Co. Iron determinations were made according to the method of Fischer and Price (23). Spin quantitations were performed as described by Aasa and VanngHrd (24).

Enzymes were transferred to "0-enriched water by concentrating the enzyme solution to 75-175 mg/ml and then diluting it IO-40-fold into buffer prepared with "0-enriched water. Nitric oxide was added by slowly bubbling NO gas through an enzyme sample in an EPR tube for approximately 3 min. Argon gas which had been purged of 0 2 by passage over a column of BASF Inc. copper catalyst was flushed continuously over the top of the sample to prevent reaction of the NO with 02. The samples were frozen by slow immersion in liquid Nz except where noted. Samples in ["OO]water and ["Olwater were made in parallel.

The enzymes were titrated with NO by preparing NO-Ar mixtures totaling 1 atm on a vacuum line. Samples of enzyme were introduced into a chamber connected to the vacuum line and allowed to equili- brate with stirring for 5 min on ice. Incubations of up to 15 min were not found to alter the results. Each sample was then transferred to an EPR tube also connected to the vacuum line and frozen in liquid N2. The fractional saturation of the enzyme nitrosyl complex was determined by double integration of the EPR spectra, and the dissolved NO concentration was obtained from standard solubility ta- bles. Data was fit to standard binding equations using a nonlinear regression program adapted from that of Bevington (25) by E. Ec- cleston, University of Minnesota.

Ternary enzyme substrate (or inhibitor) nitrosyl complexes could be made by initially adding either substrate or NO to the enzyme. The order of addition did not change the EPR spectrum of the resulting complex. The spectra shown in Figs. 1 and 2 were prepared by adding NO to pre-formed enzyme complexes with inhibitors or substrates. Fe2' .EDTA.NO complex was made by bubbling NO through an anaerobic solution of 5 mM EDTA, 2 mM Fe(NH&(SO&, and 10 mM sodium dithionite prepared in either l7O-enriched or unenriched water.

EPR spectra of intermediate spin S = Yz complexes were analyzed according to the spin Hamiltonian:

where D and E/D are zero field-splitting parameters, & is the transferred hyperfine coupling tensor of the 170-ligand, and the other parameters have their usual definitions. The hyperfine terms IF.. AFe. $j and I,. A,. S describe interaction of the electron spin with the nuclear spin of the Fe and the nuclear spin of the ligand, respectively. Since the nuclear spin of 56Fe is 0, the first term has no effect on the spectra of samples not enriched in 57Fe ( I = %). The second term results in split EPR resonances due to the I = 115 nuclear spin of l 7 0

however, the splitting is not resolved in the spectra reported here, and the effect is observed as a broadening of the signal. The term E / D is a measure of the departure of the electronic environment of the Fe from axial symmetry and can assume values between 0 and Y3 (26, 27), the extreme values representing the axial and completely rhombic cases, respectively.

In zero applied field, the spin quartet characteristic of an S = 3/2 spin system is split into two degenerate doublets by the ligands. These two energy levels are separated by an energy I A I = ZD(1 + 3(E/ D)2)K. In an applied magnetic field, the degeneracy of the doublets is removed and EPR can be observed for transitions within the doublets. For magnetic fields such that g&7 << A, the magnetic properties of each doublet can be described separately using a spin Hamiltonian with an effective spin S' = %:

= P J ' . j . H (2)

where g is the electronic g tensor. The principal components of (gz,

g,, and gz) are computed separately for each doublet. For small values of E/D, transition probability within the upper doublet is very small, and thus, EPR signals are not usually observed. In contrast, the lower doublet gives rise to an intense EPR signal with g values given

["O] Water and NO Binding by Extradwl Dioxygenases 14037

approximately by:

gz = go[2-3E/D - 3/2(E/D)'], (3) gr = go[:! + 3E/D - 3/2(E/D)'], and g, = go[l - 3(E/D)*]

These equations are valid as long as the departure from axial symmetry is not too great (E/D < 0.15), which is the case for the nitrosyl complexes of the dioxygenases (E/D < 0.1). According to these equations, if go is taken to be 2, the three principal g values will be found at g = 4.0, 4.0, and 2.0 when E/D = 0. As E/D increases, the two g values found at g = 4.0 will be split approximately equally around 4.0 while the g value at 2.0 will decrease only slightly (see Ref. 28 for further discussion). The E/D value can be calculated from the g values. For example, one of the two species observed in the EPR spectrum of the protocatechuate 4,5-dioxygenase-PCA-N0 complex exhibits resonances at g = 4.22, 3.77, and 1.98; these correspond to E/D of 0.037.

RESULTS

Water Binding in the Native Enzyme Nitrosyl Compkxes- Nitrosyl complexes of catechol 2,3-dioxygenase (Fig. 1) and protocatechuate 4,5-dioxygenase (Fig. 2) exhibit EPR spectra with resonances near g = 4 and g = 2 characteristic of an S = 3/~ spin system. Preparation of the nitrosyl complex of either enzyme in 170-enriched water results in a 0.4-0.7 millitesla

H- FIG. 1. Binding of "0-enriched water by nitrosyl com-

plexes of catechol 2,3-dioxygenase. Superimposed EPR spectra of NO complexes of catechol 2,3-dioxygenase (60 /IM) in the presence of I70-enriched (-) water and unenriched (. . . . .) water. A, native enzyme; B, ternary complex with 5 mM catechol; c, ternary complex with 2-OH-pyridine N-oxide. Measurement conditions: microwave frequency, 9.22 GHz; microwave power, 0.2 m W modulation frequency, 100 kHz; modulation amplitude, 1 millitesla ( r n T ) ; center field, 166 millitesla; field sweep, 40 millitesla; temperature, 6.0 K, relative gain and g values are shown on the figure. Insets are the g = 2 region with center field at 335 millitesla and a field sweep of 20 millitesla.

A

x 1 1,%

4.37 f B A

1

ii

x1.5

- 5 mT

26 I

lr- 4.73

FIG. 2. Binding of "0-enriched water by nitrosyl complexes of protocatechuate 4,5-dioxygenase. Superimposed EPR spectra of NO complexes of protocatechuate 4,5-dioxygenase (120 FM) in the presence of 170-enriched (-) water and unenriched ( . . . . . ) water. A , native enzyme; B, ternary complex with 5 mM PCA (spectra normalized to the g = 4.21 resonance); C, ternary complex with 5 mM 2-OH-INO. Measurement conditions are given in Fig. 1 except that the field sweep is 50 millitesla. The g = 2 region was obscured by an anomalous resonance from NO itself. This signal is particularly prevalent in buffers prepared without acetone, thus the g = 2 region is masked here but not in the spectra of Fig. 1.

broadening of the EPR resonances as shown in Figs. LA and 2A and summarized in Table I. The dilution of the [170]water with [160]water which is incurred during preparation of the samples results in a final solution containing approximately 40 atom ?6 enrichment. Thus, the maximal broadening due to 'IO hyperfine interaction is somewhat greater'than that observed. Broadening is apparent in at least one and probably all of the resonances for each of the three major S = Yz type species of the catechol 2,3-dioxygenase nitrosyl complex (the 3 species are better resolved in the data shown in Fig. 6). Although the g = 2 resonances associated with these species are superimposed and partially obscured by the anomalous resonance below g = 2 (an artifact of the NO itself), one or more of the resonances is clearly broadened (Fig. lA, inset). Likewise, broadening is apparent in each g value of the single species exhibited by the protocatechuate 4,5-dioxygenase-N0 complex (Fig. 2%). Transferred hyperfine interaction of this type is observed only when there is a bond between the ligand with a nonzero nuclear spin, such as 170 (I = "z.), and the Fe (29). Thus, this data constitutes direct evidence for a water ligand to the Fe in the nitrosyl complex of each enzyme.

Nitric Oxide Binding to the Active Site Fe-Although Fe ligation of NO has not been demonstrated directly in dioxygenase complexes, the dramatic redistribution of electron

14038 ['70] Water and NO Binding by Extradiol Dioxygenases

TABLE I Water ligation by nitrosyl adducts of extradiol dioxygenases

Enzyme or Fe chelate &' Line width" Water Km, Kr, oI

liga- value IEOH, 1 7 0 ~ ~ tion K H b

Protocatechuate 4,5- &oxygenase

(E/D = 0.015) Native

Substrates Protocatechuic acid (E/D = 0.064)

(E/D = 0.037

3,4-(OH)2-phenyl- sulfonate

(E/D = 0.064)

(E/D = 0.036)

Substrate analogs 2-OH-INOf (E/D = 0.044)

6-OH-NNO' (E/D = 0.036)

Protocatechualdehyde (E/D = 0.044)

(E/D = 0.022)

Inhibitors 3-OH-benzoic acid (E/D = 0.013)

4-OH-benzoic acid (E/D = 0.030)

(E/D = 0.021)

3-NH2-benzoic acid (E/D = 0.016)

4-NH2-benzoic acid (E/D = 0.007)

millitesh mM

4.09 2.3 2.7 Yes 3.91 2.45 3.1

4.37 2.8 2.8 No K,,, = 0.08 3.57 2.7 2.8 4.21 2.6 2.7 No 3.78 2.7 2.7

4.37 3.65 3.7 No ND' 3.61 5.6 5.6 4.21 d*e No 3.78 d,e

4.26 3.2 3.2 NO K, = 0.01 3.73 4.25 4.25

4.21 3.60 3.60 NO K% = 0.25 3.72 4.00 4.05

4.25 4.5 4.5 NO K, = 1.9 3.75 3.5 3.5 4.18 dse No 3.92 d,e

4.07 5.15 4.5 Yes K% = 3.4 3.93 5.0 5.0

4.17 2.1 2.3 Yes K, = 1.0 3.82 2.1 2.55 4.12 d*h Yes 3.87 d.h

4.10 d,h Yes K, = 13 3.90 d,h

4.04 NO K% = 5.0 3.96 6.45

3-F-4-OH-benzoic acid (E/D = 0.028) 4.17 2.0

3.83 2.45 (E/D = 0.014) 4.09

3.91 3-C1-4-OH-benzoic

acid (E/D = 0.028) 4.17 2.1

3.83 2.2 (E/D = 0.013) 4.08

3.92 3-I-4-OH-benzoic acid (E/D = 0.025) 4.15 2.45

3.84 2.4 (E/D = 0.009) 4.05

3.95

-

-

a Measured as full width at half-height or peak to peak * K, = Concentration required to reduce Vi by one-third in an assay with [PCA] = 0.080 mM.

\

e ND, not determined.

e Broadening is not observed. '2-OH-IN0 = 2-hydroxyisonicotinic acid-N-oxide. 6-OH-NNO = 6-hydroxynicotinic acid-N-oxide.

h Broadening is observed. ' Minor line shape changes relative to the spectrum of native enzyme are observed.

Overlap of resonances prevents line width determination.

6.4

2.55 Yes K, = 0.20

ah Yes 3.0

d. h

2.4 Yes K, = 0.015

d,h Yes 2.6

d,h

2.85 Yes K,= 0.05

d,h Yes 3.0

d h

millitesln mM

3-OCH3-4-OH-benzoic acid

(El0 = 0.029) 4.17 2.15 2.55 Yes K, = 0.25 3.82 2.25 2.65

(EID = 0.0141 4.08 d,h Yes . ,

3.94 d,h

3-NO2-4-OH-benzoic acid

(E/D = 0.025) 4.14 2.4 2.8 Yes K, = 0.25 3.84 2.6 3.1

3-NHz-4-OH-benzoic acid

(EID = 0.015) 4.09 3.35 4.15 Yes KG = 2.0 3.91 3.1 3.85

3-OH-4-NH,-benzoic acid

(E/D = 0.030)

(E/D = 0.012)

Catechol 2,3-&oxygenase Native

(E/D = 0.029)

(E/D = 0.021)

(E/D = 0.10)

4.17 4.2 4.0 NO K% = 2.0 3.82 3.6 3.7 4.07 2.65 3.5 No 3.93 3.7 4.0

4.18 d,h Yes 3.83 2.2 2.5 1.99 1.9 2.5 4.14 d,h Yes 3.90 2.6 3.2 4.09 3.97 ah

d,h Yes

Substrates Catechol (EID = 0.005) 4.04

3.98 d+ No K,,, = 0.015 d,e

2.00 1.60 1.60 4-CH3-catechol (E/D = 0.010) 4.08 dse No K,,, = 0.050

3.96 d*e

2.00 1.55 1.55 Substrate analog

2-OH-pyridine N-oxide (E/D = 0.010) 4.08 3.80 3.80 NO KI= 0.006

3.96 3.60 3.60 2.00 1.75 1.75

(E/D = 0.055) 4.30 2.80 2.80 No Inhibitors

2-OH-benzyl alcohol (E/D = 0.035) 4.20 3.1 3.1 NO KI = 3.95

3.79 3.25 3.25 1.99 1.55 1.53

tive enzyme)'

tive enzyme)'

tive enzyme)'

2-Br-phenol (Same as na- Yes Kr = 0.23

2-F-phenol (Same as na- Yes Kr = 0.75

2-OCH3-phenol (Same as na- Yes KI = 0.26

Fe(I1)-EDTA-NO (E/D = 0.016) 4.10 3.90 3.90 No ND'

3.90 4.90 4.90 2.00 2.50 2.50

vidth of the EPR resonance.

["O] Water and NO Bindin I I

- 4 mT

H" FIG. 3. EPR spectra of the nitrosyl complex of "Fe-en-

riched catechol 2,3-dioxygenase. Superimposed spectra of 57Fe- enriched (-) and unenriched (- - - - -) catechol 2,3-&oxygenase. Solvent and measurement conditions were as in Fig. 1. mT, millitesla.

density required to allow the Fez+ center to assume an S = 3/~ spin state, the characteristic EPR and Mossbauer spectra (12) which result, and the facile reversibility of the binding process (see below) leave little doubt that NO is a ligand. Several inorganic model complexes of Fez+ will bind NO to generate species exhibiting S = 3 /~ EPR spectra. Crystallo- graphic studies of some of these complexes, Fe(NO)salen, for example (30), have shown that the nitrogen of NO is coordinated directly to the iron.

It can be readily demonstrated that the S = %-type EPR signal derives from the Fe through comparison of the NO complexes of 67Fe-enriched enzyme and unenriched enzyme. Hyperfine interaction of the electronic spin with the I = Yi nuclear ground state of 57Fe results in approximately 0.6 millitesla broadening of the spectral features near g = 4 of the 57Fe-enriched protocatechuate 4,5-dioxygenase nitrosyl complex (data not shown) and the catechol 2,3-dioxygenase- substrate nitrosyl complex shown in Fig. 3. The broadening in this spectral region is equivocal, since it is relatively small and, more importantly, an authentic comparison with unenriched enzyme is impossible. The resonance in the g = 2 region for the protocatechuate 4,5-dioxygenase complex is partially obscured by the anomalous NO signal, but it also appears to be broadened. The g = 2 region in the spectrum of the NO adduct of the catechol 2,3-dioxygenase-catechol complex is not seriously obscured; the resonance is broadened by approximately 1.4 millitesla resulting in a doubling of the line width (Fig. 3, inset). Broadening of this magnitude and the appearance of a resolved doublet are clearly indicative of 57Fe hyperfine coupling?

Other Potential Sources of Spectral Broadening-It is con- ceivable that the observed 170 hyperfine broadening could be caused by exchange of 170 into the NO from water (31). It is clear, however, that this process does not occur spontaneously

* The hyperfine coupling constant, A,, can be calculated directly from this spectrum since the hyperfine interaction results in resolved resonances:

A, = gzPeaH

where AH is the splitting (1.4 k 1 millitesla) between the resonances. The calculated value, A, =: 39 f 3 MHz (= A k since (Bo is isotropic to within a few per cent) is slightly larger than the A , = A , values measured by Mossbauer spectroscopy for this complex (Aoz = AW = 34 f 2 MHz) and the protocatechuate 4,5-dioxygenase-PCA-N0 complex (Aox = A , = 34 & 2 MHz) (12).

g by Extradiol Dioxygenases 14039

under the conditions utilized here, since the spectrum of the Fe2+.EDTA.N0 complex, which exhibits EPR and Moss- bauer spectral features nearly identical to those of protocatechuate 4,5-dioxygenase (12, 17)) is not broadened for samples prepared in [170]water (Table I). There are examples of enzymes from the denitrification pathways of bacteria which can catalyze the NO-water-oxygen exchange (31). Such exchange is very unlikely in the case of the dioxygenases because spectra of the enzyme-substrate-NO complexes prepared by simply adding substrate to the enzyme-NO complexes are not broadened (see below).

Preparation of the enzyme nitrosyl complexes in 180-enriched water, the major contaminant in the 170-enriched water, does not result in any detectable broadening, as ex- pected, since the nuclear spin of l80 is zero. There is also no detectable shift in the g values which could result from a heavy atom effect of "0 commonly observed in NMR spectroscopy. Moreover, the spectra of samples prepared in 170- enriched water exhibit no apparent shift in E/D value as observed previously for protocatechuate 3,440xygenase and some of its complexes prepared in 170-enriched water (4). Such shifts can result from either the heavy atom effect or a weak second order interaction which is observed only when the quadrupole moment of the 170 is significant relative to the Zeeman interaction (32).

Enzyme-Substrate-Nitrosyl Complexes-Anaerobic addition of substrates to nitrosyl complexes of catechol 2,3-dioxygenase or protocatechuate 4,5-dioxygenase or addition of NO to the substrate complexes of the enzymes results in dramatic changes in the EPR spectra (Figs. 1B and 2B, and Table I). The nature of the change is distinctly different for each enzyme, but all of the resulting species retain an electronic spin of S = 3/~. EPR spectra of the resulting complexes are not detectably broadened when the samples are prepared in l7O-enriched water. This result is most easily observed in the spectrum of the catechol 2,3-dioxygenase-catechol-N0 complex (Fig. 1B) which consists of one dominant species and a minor species with a similar E/D value. No broadening is detected in any of the resonances. In the more complicated spectrum of the protocatechuate 4,5-dioxygenase-PCA-N0 complex (Fig. ZB), the two dominant species with E/D values of 0.037 and 0.064 do not appear to be broadened, although there is a slight change in the distribution of the species resulting from sample preparation procedures. When each resonance is normalized separately to the same signal inten- sity, no broadening is detected.

Several alternate substrates are known for both enzymes. As shown in Table I, none of the ternary enzyme complexes formed with these substrates and NO show any hyperfine broadening. Thus, it is likely that water is excluded from the Fe ligation by substrate binding. Direct ligation of the substrate by the Fe in the water binding site would most directly account for this result, however, conformational changes and steric effects of the substrate binding near the iron could also be invoked.

Enzyme-Inhibitor-Nitrosyl Complexes-Substitutions of any kind for the hydroxyl groups of the dioxygenase substrates result in compounds which are not turned, over and frequently serve as effective inhibitors. The EPR spectra of the nitrosyl derivatives of many of these enzyme inhibitor complexes are distinct from those of the native enzyme or the enzyme- substrate complexes. Some of these spectra are sufficiently sharp to observe the presence or absence of 170-induced hyperfine broadening. The binding of water to the Fe in these complexes was assessed and the results are shown in Table I. In contrast to the substrate complexes, several of the inhibitor

14040 Water and NO Binding by Extradiol Dwxygenases

nitrosyl complexes do exhibit broadening when prepared in 170-enriched water, showing that water is bound. In the case of protocatechuate 4,5-dioxygenase, water is bound in the nitrosyl complexes of both 3 and 4 mono-hydroxyl substituted benzoates; the spectrum of the ternary enzyme complex containing the latter is shown in Fig. &. The analogous inhibitor for catechol 2,3-dioxygenase, phenol, could not be tested because it is effectively not bound by the enzyme. Other inhibitors for protocatechuate 4,5-dioxygenase with a hydroxyl group in the 4-position and NH,, OCH3, C1, F, I, or NOz (Fig. 4B) in the 3-position also form ternary complexes which exhibit unique and broadened EPR spectra. However, inhibitors with an amino group in the 4-position and either a proton or hydroxyl id the 3-position fail to show broadening. In the case of catechol 2,3-dioxygenase, all disubstituted, mono-hydroxyl inhibitors tested show broadening irrespective of the chemical nature or size of the group in the adjacent ring position. Inhibitors with two adjacent hydroxyl groups, including 2-hydroxybenzyl alcohol, fail to show broadening.

N-oxide analogs of PCA are strong inhibitors of the protocatechuate 4,5-dioxygenase reaction. Both 2-hydroxyisonicotinic acid-N-oxide (2-OH-INO) and 6-hydroxynicotinic acid- N-oxide (6-OH-NNO) are observed to eliminate the broadening in the EPR spectra of their ternary nitrosyl-enzyme complexes prepared in 170-enriched water (Fig. 2C and Table I). Likewise, the analogous catechol 2,3-dioxygenase inhibitor, 2-OH-pyridine N-oxide eliminates the broadening in the EPR spectrum of the ternary enzyme complex (Fig. IC).

Inhibitor Binding Constants-All of the inhibitors shown in Table I for catechol 2,3-dioxygenase are competitive with catechol so that Kr values could be readily obtained by measurement of the initial velocity of oxygen uptake in the presence of fixed inhibitor concentrations and variable substrate concentrations. The KI values obtained (Table I) are, in theory, equal to the K D value for the inhibitors since the reaction appears to be ordered. The affinity of the protocatechuate 4,5-dioxygenase inhibitors are more difficult to measure because the substrate turnover reaction appears to be biphasic (33). An initial fast phase is observed which is dependent upon the preincubation time of the enzyme at the dilution used for the assay as well as the nature and concen-

I H I

FIG. 4. Binding of l'0-enriched water by protocatechuate 4,5-dioxygenase-inhibitor-N0 complexes. Superimposed spectra of NO complexes of protocatechuate 4,5-dioxygenase in the presence of 170-enriched (-) water and unenriched (. . . . .) water. A, ternary complex with 4-OH-benzoate; B, ternary complex with 3- N02-4-OH benzoate. Measurement conditions were the same as in Fig. 2. mT, millitesla.

tration of the inhibitor present. All of the aspects of this unusual reaction are not yet understood, but it is clear that the Kr of most inhibitors is different in each phase. Thus initial velocity measurements give different values for Kr depending upon the response time of the technique used to make the measurements. The K, values (concentration of inhibitor required to decrease the initial velocity by one-third for a substrate concentration equal to its K,) for the inhibitors shown in Table I are provided so that the relative affinity can be roughly assessed. In the case of a simple competitive or uncompetitive inhibitor, K, would be equal to the Kr. The measurements were made with an oxygen electrode which records predominantly the slower phase kinetics; these are more reflective of EPR sample preparation times.

The EPR spectral data for the inhibitor complexes shown in Table I was measured for inhibitor concentrations at least 10-fold greater than the Kr or K, values of each inhibitor. Nevertheless, some of the inhibitors for each enzyme fail to alter the EPR spectrum significantly from that of nitrosyl complex of native enzyme. These inhibitors include 3-OH- benzoic acid and 3-NHz-4-OH-benzoic acid in the case of protocatechuate 4,5-dioxygenase and all of the 2-substituted phenols for catechol 2,3-dioxygenase. The EPR spectrum of each of these inhibitor complexes is broadened by "0 hyperfine interaction to the same extent as unliganded native enzyme. These results suggest that it may be possible for some inhibitors to bind near the Fe but not disrupt the ligand structure.

Nitric Oxide Binding Constants-Double integration of the EPR spectrum of the nitrosyl complex of native protocatechuate 4,5-dioxygenase prepared by saturating the sample solution with NO has shown that the S = 3/~ type spectrum accounts for nearly all of the Fe2+ present and is proportional to the enzymatic activity observed (12). Lower concentrations of NO show less development of the S = Y 2 spectrum for both dioxygenases, thus providing a method to determine the affinity of the enzymes for NO. The NO binding reaction appears to be fully reversible for each enzyme. Titrations of the enzymes with NO in the absence and presence of saturating substrate are shown in Fig. 5, A and B, respectively. Proto- catechuate 4,5-dioxygenase binds NO with a dissociation constant of approximately 360 pM. However, in the presence of substrate the affinity is 2 orders of magnitude higher, KD 3 p ~ . Catechol 2,3-dioxygenase exhibits a similar effect except that in the absence of substrate the binding isotherm is best fit under the assumption that NO binding occurs at two independent, nonidentical sites in a ratio of approximately 1:l.Z. The higher affinity site exhibits a K D of approximately 190 p~ while the KO of the low affinity site is ~ 2 . 0 mM. Catechol dramatically increases the affinity and apparently causes all of the sites to become equivalent. The K D for NO in the substrate complex is s3.4 p ~ . The origin of the multiple KD values in the case of the native enzyme is unclear since the enzyme i s known to consist of identical subunits. It is possible that there is more than one conformation of the enzyme present or that the quaternary structure of the enzyme makes the Fe sites non-identical.

Multiple S = 3/~ Type Species-Nitrosyl complexes of native catechol 2,3-dioxygenase, the substrate complex of protocatechuate 4,5-dioxygenase, and many of the inhibitor complexes of each enzyme exhibit several S = 3/2 type species. The origin of these species is not clear. It is possible that they represent multiple NO binding sites on the Fe, a variety of enzyme conformations, or several substrate binding orienta- tions in the substrate complexes. The distribution of species in protocatechuate 4,5-dioxygenase substrate complex is rel-

["O] Water and NO Binding by Extradiol Dioxygenases 14041

t A nn 1

2oL O O 0.8

0 /

0 0

A

1.6

/o- - -

2.4 3.2 CNOI, mM

loot I

I 14 28 42 56 70

CNOl, IJM FIG, 5. Binding of NO to extradiol dioxygenases. Catechol

2,3-dioxygenase (40 FM) in 50 mM KP04 buffer, pH 7.5, plus 10% acetone (open circles) or protocatechuate 4,5-dioxygenase (60 pM) in 50 mM MOPS buffer, pH 8.2 (fiued circles), was incubated on ice for 5 min in the presence of mixtures of Ar and X0 gas to give the concentrations of dissolved NO shown. The samples were,frozen in EPR tubes, then the EPR spectra measured and doubly integrated to quantitate the fraction of enzyme in the nitrosyl complex. The lines indicate the theoretical binding curves using the KD values given in the text. A, native enzymes. The theoretical curve for catechol 2,3- dioxygenase is for two independent sites in a ratio of 1:1.2 with KD = 190 p~ and 2 mM, respectively. B, native enzymes plus 5 mM PCA or catechol for protocatechuate 4,5-&oxygenase or catechol 2,3-dioxygenase, respectively.

atively insensitive, to pH, buffer ion, and added organic solvents over a broad range (E?), although a small progressive change in EPR line shape is observed as a function of pH in the range between 6 and 7. In contrast, the species distribution of catechol 2,3-dioxygenase-N0 complex is very sensitive to all of the above environmental factors as shown in Fig. 6A. In addition, the observed line width of the catechol 2,3- dioxygenase spectrum is sensitive to freezing rate. Rapid freezing, such as by quenching the sample in cold isopentane, broadens each resonance by about 10%. The distribution of species does not depend on the NO concentration in the case of either enzyme. Fig. 6B shows that the spectrum of catechol 2,3-dioxygenase is essentially unchanged over a wide range of NO concentration and degree of saturation.

The change in the distribution of species in the catechol 2,3-dioxygenase-N0 complex with pH and solvent perturbation facilitates resolution of the species in the EPR spectrum.

3.83'v I I

FIG. 6. Solvent and nitric oxide concentration effects on the H-

catechol 2,3-dioxygenase-nitrosyl complex. Superimposed EPR spectra of 60 p~ catechol 2,3-dioxygenase plus NO. A, enzyme in 50 mM KPO, buffer at pH 6.0 containing 10% acetone (-); enzyme in 50 mM KPO, buffer at pH 9.0 containing 10% acetone (- - - - -); enzyme in 50 mM KPO, buffer at pH 6.0 containing 10% ethanol (. . . . .). B, nitrosyl complex at pH 7.5 plus 10% acetone; [NO] = 3.2 mM (. . . . .); [NO] = 3.2 p M (-). Measurement conditions were the same as those in Fig. 1. mT, millitesla.

The spectra of Fig. 6A show that the broad g, resonance centered at g = 4.16 for the sample measured at pH 7.5 actually has at least two components with the associatedg, resonances at g = 3.83 and 3.91 (E/D s 0.029 and 0.023, respectively). At pH 6 in buffer containing 10% acetone, the dominant species observed in the EPR spectrum exhibits g values at 4.09 and 3.97 (E/D = 0.01); this species/is also observed in the spectrum of the pH 7.5 sample. The latter species is unusual because the g values are not uniformly split aroundg = 4. This suggests that the Zeeman term for this species is better described as an axial g tensor with go, = 2.015 and go, 2.00. Several of the complexes listed in Table I show this same characteristic. Each of the species resolved by environmental perturbation exhibits broadened EPR spectra when the samples are prepared in 170-enriched water.

DISCUSSION

Native Enzyme and Substrate Complexes-Previous studies of extradiol dioxygenases have dealt primarily with the overall structure of the enzymes and the types of reactions which they catalyze. In this study, we have begun to examine the active site structure through the use of a probe, nitric oxide, which is thought to bind directly to the iron. We have shown that in the enzyme-NO complex at least one water is bound directly to the iron and that this water is probably displaced during substrate binding. A degree of doubt is retained in the latter finding because it is possible that the alignment of, or an extreme anisotropy in, the nuclear hyperfine coupling tensor could make the broadening undetectable along one or more of the g axes. It is very unlikely, however, that the broadening would be undetectable along all three g axes as is the case in the substrate complexes described here.3 Detection of ligand binding via hyperfine broadening is a very selective

~ ~ ~ ~~~

We have shown that for the NO adducts of the substrate complexes of each enzyme the hyperfine coupling tensor, A, is at most slightly anisotropic. Moreover, the fact that A, = A , for these complexes to within our ability to measure the value (12) strongly suggests that the hyperfine coupling and zero field splitting tensors are aligned.

14042 ["OJ Water and NO Binding by Extradiol Dioxygenuses

technique which requires that a chemical bond be formed between the interacting atoms. There is no detectable broadening due to non-bonded atoms in close proximity (29). Thus, this is the first direct demonstration of ligand binding to the active site iron of extradiol dioxygenases. If the Fe coordination of the nitrosyl complex is representative of that in the native enzyme or its oxy-complex, then the findings reported here have a dire& bearing on the mechanism of this enzyme class as discussed below.

Nitrosyl Complexes as Models for Oxygen Binding-The relevance of the NO complex as a probe for the active site structure of the native enzyme and its substrate or oxygen complexes is difficult to assess. Nevertheless, there are several indications that the reaction of NO with the enzymes is highly specific and consistent with the type of reaction which might be anticipated for molecular 02. Nitric oxide has been used extensively as a probe for the oxygen binding site in heme proteins including hemoglobin (34) and cytochrome oxidase (35). The NO-Fe bond in these systems in very strong due to the back bonding potential of the heme. In ferrous heme-NO complexes an electron is thought to be delocalized onto the NO leaving the Fe in an EPR active, S = state (36, 37). The EPR signal exhibited by these complexes is split by the 14N nucleus of the NO and also by transferred hyperfine interaction from an axial histidine ligand in some cases. For the dioxygenases, we have observed no such transferred hyperfine interactions, however, the line widths of the spectra are much greater than those observed for heme-NO complexes. The Fe-NO bond is weaker than that observed for the heme proteins. Nevertheless, the KO for NO in the presence of substrate is as low or lower than the K, for oxygen (catechol 2,3-dioxygenase, K, = 7-9 pM (15, 38); protocatechuate 4,5- dioxygenase, K, = 54-303 p~ (39,40)). The large decrease in the KD value for NO observed on substrate binding shows that the NO binding site, while not created by substrate binding, is strongly influenced by it. If NO does act as an oxygen analog, and the binding of O2 is similarly coupled to substrate binding, this may provide a mechanism for the ordering of the reaction suggested by steady state kinetic studies (38). That is, the substrate free enzyme may exhibit a KD for 0, which is too high to allow significant binding at the solution concentration of O2 ( ~ 2 8 0 pM). Substrate binding would presumably decrease the KO value for O2 into the range of the observed K, values. Unfortunately, it is not possible to test NO as a competitive inhibitor for O2 due to the sponta- neous reaction which occurs between these two molecules.

Multiple EPR Active Species-The EPR spectra of the native enzyme-NO complexes of the extradiol dioxygenases studied here consist entirely of S = 3/2 type species, but they are quite distinct. The protocatechuate 4,5-dioxygenase spectrum consists of one predominant species while that of catechol 2,3-dioxygenase is composed of at least 3 species. The 3 species are clearly resolved by the pH and solvent pertur- bations shown in Fig. 6A. Each species of each enzyme is broadened by preparation in 170-enriched water showing that water is bound to the Fe in all cases. Thus, the various species do not represent water bound versus not bound, although they could reflect different numbers of waters bound or a change in the specific Fe coordination site occupied by the water. The finding that the concentration of NO present does not greatly alter the distribution of species observed for either enzyme (Fig. 6B) makes it unlikely that the species represent multiple NO binding sites unless the sites have equal affinities for NO. Since the distribution of species in the case of catechol 2,3-dioxygenase is very sensitive to the environmental factors such as the presence of organic solvents and pH, it is more

likely that the species arise from protein conformational changes occurring between pH 6 and 7. Failure to observe major pH-dependent changes in the spectrum of the protocatechuate 4,5-dioxygenase-PCA-N0 complex, which also consists of several species, may indicate that the conformation of this enzyme is not sensitive to pH in the range tested. Alternatively, the conformation of the enzymes may only be sensitive to pH in the absence of substrate. Thus, substrate free protocatechuate 4,5-dioxygenase-nitrosyl complex may show the same sensitivity to pH as observed for the substrate free catechol 2,3-dioxygenase complex, but the various species may have very similar E/D values, and therefore, remain unresolved. In support of the latter proposal, only slight changes in line shape are observed for the catechol 2,3- dioxygenase-catechol-NO complex as a function of pH and solvent.

Substrate Analog Compkxes-The two N-oxide substrate analogs for, protocatechuate 4,5-&oxygenase shown in Table I (2-OH-IN0 and 6-OH-NNO) are not turned over despite the fact that they possess two ortho-hydroxyl groups. These analogs were proposed in a previous study (10) as transition state analogs for intradiol PCA dioxygenases since they spontaneously assume the ketonized configuration thought to be important in the mechanism of these enzymes. The two analogs apparently exclude water from the Fe coordination when bound to protocatechuate 4,5-dioxygenase. Although both analogs bind well to protocatechuate 4,5-dioxygenase, their affinity is much greater for protocatechuate 3,4-dioxygenase. The 2-OH-IN0 exhibits a much higher affinity than 6-OH-NNO for both enzymes. In the case of protocatechuate 3,4-dioxygenase, each N-oxide inhibitor excludes water from the Fe coordination in a semistable, intermediate complex, but allows water to rebind in a final, stable complex. We have proposed that the change from the intermediate complex to the final complex involves inhibitor ketonization and conver- sion from a bidentate to a monodentate Fe complex (4, 10) thus opening a ligand site for water. Apparently, no similar complex with an accessible water binding site forms with protocatechuate 4,5-dioxygenase, since no broadening from 170-enriched water is observed. The same results are observed for the nitrosyl complexes of the analogous, potent inhibitor for catechol 1,2- and 2,3-dioxygenases, 2-hydroxypyridine N- oxide.

Inhibitor Compkxes-The inhibitors of the type reported in Table I exhibit a wide range of affinities for the dioxygenases. Strong inhibitors for protocatechuate 4,5-dioxygenase must have at least one hydroxyl function located in either the 3- or $-ring position relative to a negatively charged substituent; a much stronger bond is formed if it is in the 4-position. Thus, both 3- and 4-hydroxybenzoate bind, but the 4-hydroxy analog has a higher affinity. This may suggest that the 4-OH group is important for interaction with the Fe. In some cases, such as the 3- and 4-amino benzoates, inhibitors without hydroxyl functions bind, but their affinity is very low. In general, disubstituted, mono-hydroxy analogs are bound better by protocatechuate 4,5-dioxygenase than the mono-substituted inhibitors even if the second substituent cannot form a bond to Fe. Similarly, catechol 2,3-dioxygenase requires a substituent adjacent to a hydroxyl group to form even a moderately tight complex; hence, phenol binding is too weak to measure but 2-Br- or 2-OCH3-phenol bind very well. This is probably due in part to the inductive effects of the nonhy- droxyl substituent, but since the binding affinity does not appear to follow a linear free energy series, it may also involve a binding site on the enzyme for this group.

The lack of any detectable broadening in the dominant

P7O1 Water and NO Binding by Extradiol Dioxygenases 14043

species of the EPR spectra of the NO adducts of the extradiol dioxygenase-substrate complexes suggests that the substrates displace water on binding to the enzymes. The dramatic changes in line shape engendered by the substrates further suggests that they cause major changes in the Fe environment. The putative water exclusion and observed line shape changes could most easily result from either direct binding of the substrate to the Fe via the hydroxyl functions or binding in a nearby location which would sterically hinder approach of water. We have observed here that inhibitors for the extradiol dioxygenases formed by making a substitution for the 3- hydroxyl group of PCA or one of the hydroxyl groups of catechol allow water binding to the Fe regardless of the bulk of the added substituent. This observation argues against steric exclusion of water by the hydroxyl group in this position of substrates. In the case of intradiol, protocatechuate 3,4- dioxygenase, bulky groups in the 3-position of inhibitors coordinated to the Fe via a 4-position hydroxyl group were found to exclude water from the complexes while small groups did not interfere with binding (4). However, in opposition to this argument, the ternary complexes of many of the extradiol inhibitors of this type give EPR spectra which are similar to those of the native enzyme-NO complexes in the absence of exogenous organic ligands, thus the inhibitor binding orien- tation may be different than substrates.

Comparison of Extradiol Dioxygenuses-The extradiol dioxygenases studied here have many similarities, most notably in the overall reactions which they catalyze, the requirement for Fez+, their mutual ability to bind NO to form an S = YZ type complex, and the presence of a substrate perturbable, water binding site on the Fe in the nitrosyl complex. Never- theless, the two enzymes differ in details within these broad areas of similarity. For example, the Mossbauer spectra of the Fe2+ centers exhibit different parameters (12, 16). Also, the EPR line shapes of the nitrosyl complex are markedly different and the catechol 2,3-dioxygenase substrate-NO complex exhibits a weak optical chromophore not observed for protocatechuate 4,5-dioxygenase. In addition, there are structural differences between the enzymes. For example, protocatechuate 4,540xygenase is composed of two types of subunits while catechol 2,3-dioxygenase has only one type (12, 15). These distinctions between the extradiol dioxygenases suggest that while the overall mechanism is probably very similar, there are likely to be subtle differences in its application for each enzyme.

Relationship t o the Mechanism of Intradiol Dioxygenases- Recent studies have suggested that protocatechuate 3,4-dioxygenase, and presumably other intradiol dioxygenases, have at least two sites in the Fe coordination which can be occupied by exogenous ligands (4, 41, 42). Data from our laboratory has been used to formulate a model for the protocatechuate 3,4-dioxygenase mechanism in which substrate in the ketonized conformation binds to one of these sites while the second site functions to stabilize a peroxide formed after the attack of oxygen on the substrate (4, 10). The results presented here show that the active site Fe of the extradiol dioxygenases also possesses multiple sites in its coordination for external ligands. It is likely that substrate either binds at or near two of these sites. In the case of protocatechuate 3,4-&oxygenase, a NO complex which exhibits an S = 3/2 type EPR signal can be formed if the enzyme is first chemically reduced and then exposed to NO (17). However, unlike the nitrosyl complexes of the extradiol dioxygenases described here, NO apparently excludes water from the Fe coordination: Thus it is likely

* T. P. Ikeda and J. D. Lipscomb, unpublished observation.

that the NO occupies the same site or sites as water. Water is also apparently excluded by substrates in the intradiol dioxygenases suggesting that the NO and substrate may compete for at least one coordination site on the Fe. In the case of the extradiol dioxygenases, the NO site is not destroyed or masked by substrate binding, thus it is likely that a separate site exists for small molecules such as NO, and perhaps 0,. If O2 does bind to the Fe2+, then it is likely that oxygen activation for insertion occurs by a mechanism more in line with that proposed for the initial steps for the cytochrome P-450 (43) or tryptophan 2,3-dioxygenase (44) reactions, in which inver- sion of the oxygen ground state triplet is catalyzed by delo- calization into the d orbitals of the metal. This is in marked contrast with the oxygen activation mechanism of intradiol dioxygenases which probably involves intermediate substrate and oxygen radicals (3,4).

Acknowledgments-We would like to thank Dr. Eckard Miinck for analysis of the Mossbauer spectra of catechol 2,3-dioxygenase-catechol-NO complex and Dr. E. Eccleston for adaptation of the nonlinear regression program used in this study.

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[l'o]water and nitric oxide binding by protocatechuate 4,b

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