CHARACTERIZATION OF AN OXYGENATED INTERMEDIATE IN A DIOXYGENASE REACTION BY EPR AND MÖSSBAUER SPECTROSCOPY

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CHARACTERIZATION OF AN OXYGENATED

INTERMEDIATE IN A DIOXYGENASE REACTION

BY EPR AND MÖSSBAUER SPECTROSCOPY

E. Münck, R. Zimmermann, J. Lipscomb, L. Que, W. Orme-Johnson

To cite this version:

E. Münck, R. Zimmermann, J. Lipscomb, L. Que, W. Orme-Johnson. CHARACTERIZATION

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JOURNAL DE PHYSIQUE Colloque C6, supplement au n° 12, Tome 37, Decembre 1976, page C6-203

CHARACTERIZATION OF AN OXYGENATED INTERMEDIATE

IN A DIOXYGENASE REACTION BY EPR

AND MOSSBAUER SPECTROSCOPY

E. MUNCK, R. ZIMMERMANN (*), J. D. LIPSCOMB and L. QUE, Jr. Freshwater Biological Institute, University of Minnesota, Navarre, MN 55392, U. S. A.

and

W. H. ORME-JOHNSON

Department of Biochemistry, University of Wisconsin, Madison, WI 53706, U. S. A.

Résumé. — Les dioxygénases sont des enzymes capables de couper la composition aromatique

par l'installation d'oxygène. Un état intermédiaire dans la réaction catalytique est le triple complexe d'enzyme, de substrat et d'oxygène. Tandis que les complexes semblables ont été étudiés large-ment pour des protéines hèmes, on n'a pas fait beaucoup de publications pour les états intermé-diaires des réactions de dioxygénases. Ici nous caractérisons le complexe de protocatéchuate 3,4-dioxygénase avec 3,4-dihydroxyphénylpropionate et O2 par l'application de EPR et la spec-troscopie Môssbauer. Le complexe avec oxygène a des signaux EPR près de g = 6,7 et 5,3. Ces signaux résultent d'une doublette Kramer excitée. Les données Môssbauer et EPR montrent que le fer est dans un état de spin-fort ferrique, caractérisé par une valeur grande et négative D de l'Ha-miltonien des spin, D — — 2 cm-1. Une telle espèce n'est pas observée jusqu'au présent dans les systèmes biologiques.

Abstract. — Dioxygenases are enzymes that cleave aromatic compounds by oxygen insertion. An important intermediate in the catalytic reaction is a ternary complex of enzyme, substrate, and oxygen. While such complexes ha\e been studied extensively for heme proteins, very little work has been published for such intermediates in dioxygenase reactions. Here we report the characterization of a complex of protocatechuate 3,4-dioxygenase with 3,4-dihydroxyphenylpro-pionate and O2 using EPR and Mossbauer spectroscopy. The oxygenated complex yields EPR signals at g = 6.7 and 5.3 resulting from an excited state Kramers doublet. The Mossbauer and EPR data establish the iron to be in a high-spin ferric state characterized by a large and negative zero-field splitting, D 2; —2 cm-1. Such a species so far has not been observed in biological systems.

1. Introduction. — Many dioxygenases are iron proteins that play a crucial role in the degradation of aromatic compounds such as phenols, fiavanoids, alkaloids, and lignin (the major structural component of wood). These enzymes catalyze the cleavage of aro-matic rings by insertion of two oxygen atoms, both derived from the same oxygen molecule. Protocate-chuate 3,4-dioxygenase (3,4 PCase) from

Pseudo-monas aeruginosa catalyzes the cleavage of

3,4-dihydro-xybenzoate (protocatechuate) to fl-caxboxy-cis,cis-muconate (Fig. 1).

Coo-

coo-FIG. 1. — 3,4 PCase catalyzes the cleavage of protocatechuate to j8-carboxy-cw,cw-muconate by oxygen insertion. (*) On leave from the University Erlangen-Nurnberg, D852, Erlangen, Germany.

In the past decade this enzyme has been studied extensively by Hayaishi and coworkers [1]. Molecular weights of 700,000 and 660,000 have been estimated for the crystalline enzyme by sedimentation equilibrium and total amino acid composition [1]. The enzyme consists reportedly of eight identical subunits, each containing one iron atom. In the course of this work we have reinvestigated the subunit structure of 3,4 PCase and found that each subunit is a tetramer consisting of two different polypeptides in an <x2 P2 structure [2].

The native enzyme displays an optical absorp-tion spectrum characterized by a broad band near 450 nm [1] which shifts to 480 nm upon anaerobic addi-tion of substrate [3]. Addiaddi-tion of molecular oxygen yields a short-lived intermediate which has been associated with an enzyme-substrate-oxygen com-plex [4]. While the nature of such comcom-plexes has been extensively investigated for heme proteins, very little work has been published for such intermediates in dioxygenase reactions. In this communication we report the characterization of the ternary complex of

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C6-204 E. MUNCK, R. ZIMMERMANN, J. D. LIPSCOMB, L. QUE, JR. AND W. H. ORMEJOHNSON

3,4 PCase with 3,4-dihydroxyphenylpropionate and 0, by electron paramagnetic resonance (EPR) and Moss- bauer spectroscopy. Our work has yielded some unique spectral parameters which should serve as useful guide- lines for the design of inorganic model complexes for this enzyme. In this paper we will concern ourselves with the oxygenated intermediate ; we have reported Mossbauer and EPR results on the native enzyme and its reduced form elsewhere (L. Que et al., submitted for publication).

2. Methods. - Details of the purification procedure

have been described previously (L. Que et al., submitted for publication). The EPR samples for studying the decay of the oxygenated intermediate were made as follows : 3,4 PCase, 3,4-dihydroxyphenylpropionate,

and buffer solution (Tris-C1 buffer at pH 8.5) saturated with 0, were incubated in an EPR tube at 6 OC and observed in a Cary 14 spectrophotometer with the EPR tube positioned in the light beam. When the appro- priate change in the optical absorption spectrum had occurred, the sample was frozen by rapid submersion in a stirred isopentane bath at 130 K ; the samples freeze within 1-2 seconds with this procedure.

The Mossbauer samples were made by injecting substrate into enzyme solution saturated with 0, at 3 atmospheres at 6 OC and quickly freezing the mixture. EPR spectroscopy was performed at X-band on a Varian E-9 spectrometer in the temperature range from 6-40 K using helium boil-off gas as a coolant. Tempe- ratures were measured with a calibrated carbon resistor kept below the sample in the gas stream. The Moss- bauer data were taken on a constant acceleration spectrometer. Our velocity transducer (Elscint MVT-3) was quite sensitive to the presence of small magnetic fields (10 gauss) ; therefore it was enclosed in an iron box.

3. Results. - The iron atoms in native 3.4 PCase are in the high-spin ferric state. Within the resolution of EPR and Mossbauer spectroscopy they are indistin- guishable (L. Que et al., submitted for publication). Upon binding of substrates complex EPR and Moss- bauer spectra are observed (for an EPR spectrum see Fig. 2C below). Although the details of these spectra remain to be explored, it is clear that the material is still high-spin ferric and that substrate binding is associated with changes in the electronic structure of the irons. Addition of oxygen to a mixture of enzyme and substrate results in an intermediate with a lifetime of only a few milliseconds. Fujisawa et al. [4], however,

have reported optical and kinetic data for a fairly long- lived oxygenated intermediate using the slow substrate

3,4-dihydroxyphenylpropionate. This complex decays, at 6 OC, with a half-life of about 4 minutes.

EPR spectra taken on this oxygenated intermediate, prepared as described above, are displayed in figure 2. The upper trace shows an EPR spectrum of the intermediate after steady state conditions had been achieved

FIG. 2. - EPR spectra of the ternary complex of 3,4 PCase with 3,4-dihydroxyphenylpropionate and 0 2 . Conditions :

T = 12.3 K ; 9.196 GHz, 1 mW ; modulation amplitude, 10 gauss ; sweep rate, 1 000 gauss per min ; receiver gain, 3 200 in the low-field region, 160 in the g = 4.3 region in (A) and 63 in (B) and (C). The magnetic field increases linearly to the right and selected values of the frequency-to-field ratio (g-value) are given or. the abscissa. A. The samples was frozen after steady-state conditions had been achieved. B. The sample was frozen after the ternary complex had decayed for one half-life (4 min.) as monitored by optical spectroscopy. C. The sample was frozen after the ternary complex had decayed for

four half-lives.

(the slow step in the catalytic cycle of 3,4 PCase is the decay of the oxygenated complex, i. e. product for- mation ; hence under steady state conditions we attain the maximum concentration of oxygenated material). The most prominent features in figure 2A are strong resonances at g = 6.7 and 5.3 and a weaker resonance at g = 6 (I). These resonances are only observed in the

presence of 0,. Resonances at g = 6.7 and 5.3 can result from a high-spin ferric system in an environment of almost axial symmetry. Figure 3 shows a Mossbauer spectrum of a sample prepared in a manner similar to the EPR sample for which the spectrum in figure 2A

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CHARACZI'ERIZATION OF AN OXYGENATED INTERMEDIATE IN A DIOXYGENASE REACTION BY EPR C6-205

I

I I I I I I I I I I I spin ferric systems ( S = 512) are described by the spin

FIG. 3.

-

Mossbauer spectrum of the ternary complex of 3,4 PCase with 3,4-dihydroxyphenylpropionate and 0 2 taken at 4.2 K in a 600 gauss parallel field. The prominent six-line pattern results from the ground Kramers doublet of the ter- nary complex. The residual absorption (20 %) is due to species which give rise to EPR signals at g N 9 and g = 4.3. The arrows indicate where the nuclear Am = 0 transitions of the

Mossbauer spectrum associated with the & 312 Kramers doublet should occur. Computer simulations indicate that the f 312 dou-

blet is less than 6 % populated at 4.2 K.

was obtained. The Mossbauer spectrum was taken at 4.2 K in a field of 600 gauss applied parallel to the y-radiation. The intensities of the six prominent lines did not change when the field was applied perpen- dicular to the y-radiation. This observation implies that the Mossbauer spectrum results from a Kramers doublet for which g,

-

gy

<

g, ; such doublets yield either no EPR signal (if g, = gy = 0) or only extre- mely weak signals. On the other hand, a doublet with g-values at 6.7 and 5 3 has to yield a Mossbauer spectrum with intensities depending quite sensitively on the direction of the applied field (see for instance Chapter 4, and Fig. 4 of [5]). Thus, the EPR spectrum in figure 2A

FIG. 4.

-

Energy level diagram of an S = 512 system according to eq. (1) with D < 0, t = 0.03, and go = 2.0. The effective

g-values computed from eq. (1) are given for each doublet.

and the Mossbauer spectrum of figure 3 result from different Kramers doublets. These observations can be reconciled if we assume a high-spin ferric system with a large and negative zero-field splitting.

Commonly the EPR and Mossbauer spectra of high-

Hamiltonian

All quantities in eq. (1) have their conventional mean- ing (for a discussion see for instance [5]). Figure 4 shows an energy level diagram of the electronic states for D < 0 and 1 = 0.03. We have labeled each Kra- mers doublet with quantum numbers pertinent to axial symmetry, i. e. 1 = 0. For 1 = 0.03 the doublets are still essentially pure

+

512, f 312, and f 112 states. Likewise the energy separations are practically (to within 0.1

%)

4 D and 2 D, respectively. The effective g-values of each doublet are given in figure 4 also. Our data can be explained on the basis of the diagram in figure 4, using D

--

-

2 cm-l.

At 4.2 K only the

+

512 doublet is populated. This doublet is EPR-silent and yields a six-line Mossbauer spectrum with intensities independent of the direction of the applied field. From the magnetic splitting we obtain the saturation field,

Furthermore,

and

(relative to Fe metal). These values are typical for high-spin ferric compounds.

Figure 4 predicts the EPR signals to arise from the upper Kramers doublet. We can describe the temperature dependence of the g = 6.7 resonance by the function

The exponential function in eq. (2) gives the population of the

+

112 doublet (note that D

<

0) ; the 1/T dependence results from the population difference within the

+

112 doublet. Plotted as a function of temperature I(D, T ) has a maximum at T = 4.17 D if D is measured in units of

kT.

Thus, by measuring the intensity of the g = 6.7 resonance for different tempe- ratures, the zero-field splitting parameter D can be determined. Figure 5 shows the results of such an experi- ment ; the solid line was generated from eq. (2) using

D = -

1.8 cm-l. From our EPR data we cannot

determine D to better than

+

0.4 cm-I. The margins of error, however, can be reduced if the Mossbauer data are taken into account.

The

5

312 doublet is predicted to have g-values at

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C6-206 E. MUNCK, R. ZIMMERMANN, J. D. LIPSCOMB, L. 'QUE, JR. AND W:H. ORME-JOHNSON

P

4 8 2 16 2 0 2 4 T E M P E R A T U R E K 10- 8 S I G N A L I N T E N S I T Y 6 4 #

FIG. 5. - The area (amplitude times width at half height) under the g = 6.7 resonance is plotted as a function of temperature. A representative spectrum is shown in figure 2A. The solid line

was generated from eq. (2) using D = - 1.8 cm-1.

p

computed g-values using the procedure described by Aasa and Vangaard [6] ; within the experimental uncertainties the g = 6 resonance has an intensity compatible with assigning it to the f 312 doublet. Within the framework of eq. (1) we can compute the Mossbauer spectrum associated with the

+

3/2 doublet. This spectrum will have the same magnetic pattern as that associated with the

+

512 doublet ; its magnetic splitting, however, will be scaled down by the factor

g,(+ 312 state)/gz(+ 512 state) = 6/10. Most conspi- cuously this doublet would yield two absorption lines (the nuclear Am = 0 transitions) at

+

3.3 mm/s and

-

2.4 mm/s (indicated by the arrows in Fig. 3). Obviously, these lines are not present in the 4.2 K spectrum. We conclude that at 4.2 K the f 312 doublet is popuIated less than 6

%,

a judgement based on computer simulations. This implies that

I

D

I

> 1.8 cm-l. Taken together, the EPR and Mossbauer data suggest D = (- 2

1

0.2) cm-l (,).

So far we have shown that our observations are compatible with-the energy level diagram in figure 4. If the EPR and Mossbauer spectra described here do indeed arise from the oxygenated intermediate the time course of the g = 6.7 resonance must parallel the decay of the optical spectrum as observed by Fujisawa

et al. [4]. Figure 2 shows three representative EPR

(2) We have attempted to get more information about the

middle doublet by taking a Mossbauer spectrum at 12 K. We have observed increasing absorption at velocities where the lines of the f 312 doublet Mossbauer spectrum should appear. However, quantitation of the data is virtually impossible. As can be seen from figure 2A not all irons the sample are in the desired oxygenated state. There is an 'appreciable amount of absorption arotmd g = 9 and g = 4.3. Absorption due to this species can be discerned also in the Mossbauer spectrum in figure 3 ; we have estimated that approximately 80 % of the irons are in the desired state..

spectra taken under steady state conditions (A), after

one half-life (4 min, at 6 OC, B), and after four half- lives (C), respectively.! These spectra show that the decay of the resonances' at g = 6.7, 5.3, and 6 corre- lates with the decay of the oxy complex. Since the reaction mixture was 0, limited, and since substrate binding is rapid, the EPR spectrum shown in figure 2C should reflect an enzyme-substrate complex. Indeed, the same EPR spectrum is observed when enzyme and substrate are mixed anaerobically. Finally, we have studied the complex of 3,4 PCase with product, j3-carboxy-cis,cis-muconate ; the EPR spectrum is quite different from that shown in figure 2 ~ :

4. Discussion.

-

The work reported here is a small

part of a research project on 3,4 PCase which involves the skills of many researchers from quite different disciplines. On the basis of this research we have recently proposed a mechanism for 3,4 PCase cata- lysis [7]. In the present paper we have focused entirely on the nature of the ternary oxy complex. Two results of this investigation are of special interest. First, the oxygenated intermediate in the. catalytic cycle of 3,4 PCase is a complex involving the ferric ion and, secondly, this complex is characterized by a large and negative zero-field splitting. A system with a large and negative zero-field splitting so far has not been observed for an iron protein. The parameters reported here are unique and should therefore serve as useful guides for the design of model complexes mimicking the active site of 3,4 PCase. Furthermore, the data show clearly that the electronic state of the iron changes drastically upon oxygen binding. Although we do not fully understand the EPR spectra of the enzyme-substrate complexes (there are always at least two species no matter what substrate is used) one conclusion can be drawn : the species observed are characterized by large and positive zero-field splittings, in contrast to the ternary oxy complex.

The spectroscopic parameters obtained for the oxy complex differ from those found for other oxygen complexes and they suggest a new mode of oxygen binding in biological systems. Our data suggest, but do not prove, that the oxygen molecule may coordinate directly to the iron. The details of binding, however, need further elucidation. Resonance Raman studies and ENDOR experiments should give further clues to the nature of this catalytically important intermediate.

Acknowledgments.

-

This work was supported by

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CHARACTERIZATION OF AN OXYGENATED INTERMEDIATE IN A DIOXYGENASE REACTION BY EPR C6-207

References

[I] FUJISAWA, H. and HAYAISHI, O., 5. Biol. Chem. 243 (1968) [5] MUNCK, E, and CHAMPION, P. M., J. Physique Colloq. 12

2673-2681. (1974) C6-33-46.

121 LIPSCOMB, J,, HOWARD, J., LORSBACH, T. and WOOD, J., ~ ~ d[61 A&A,.R. and . ~ A N N G A R D , T-7 J- Ma@. Res. 19 (1975) 308-315.

Proc. 35 (1976) 1536. [7] WOOD, 3. M., LIPSCOMB, J. D., QUE, L., Jr., STEPHENS, R. S., ORME-JOHNSON, W. H., MUNCK, E., RWLEY, W. P., [3] FUJISAWA, H., UYEDA, M., KOJIMA, T., NOZAKI, M. and _DIZIKES,_{L., CHEH,}_{A., FRANCIA,}_{M., FRICK,}_{T., ZIM-}

HAYAISHI, O., J. Biol. Chem. 247 (1972) 4414-4421. _MERMANN, _{R. and HOWARD,}_{J., Biological Aspects}_of [4] FUJISAWA, H., HIROMI, K., UYEDA, M., OKUNO, S., Inorganic Chemistry (Proceedings of the International NOZAKI, M. and HAYAISHI, O., J. Biol. Chem. 247 (1972) Symposium) (in press), David Dolphin, editor (Wiley-