Spectroscopic Studies of the Catechol Dioxygenases Lawrence Que, Jr. University of Minnesota, Minneapolis MN 55455 The catechol dioxygenases are bacterial iron-containing enzymes that catalyze the oxidative cleavage of catechols (1).These enzymes serve as a component of.nature's mechanism for degrading aromatic compounds in the environment (2). The elucidation of the novel substrate activation mechanism (3) for this reaction has come about as a result of spectroscopic investigations on the enzymes coupled with comparative studies of model compounds. Central to these studies has been the phenolate-to-Fe(II1) charge transfer transition which persists in all the enzyme complexes and determines the UV-visible, resonance Raman, and paramagnetic NMR properties of the iron complex in these enzymes
is in progress (9). The catechol dioxvzenases are neither heme rote ins nor iron suiiur proteins;;he iron center is simply cobrdinated to ligands on the polypeptide chain. EPR and Mosshauer sper-
(4).
The catechol dioxygenases catalyze the reaction shown below
In the process of cleaving the Cl-C2 bond of the catechol, the enzyme incorporates the elements of dioxygen into the product (51,thus the term dioxygenase for describing the enzyme. The two best-studied examples are catechol1,2-dioxygenase (CTD) and protocatechuate(3,4-dihydroxybenzoate) 3,4-dioxygenase (PCD). CTD, from Pseudomonas aruilla grown on benzoate as its sole carbon source, has a molecular weight of 63,000with two nonidentical subunits (aBFe) (5). PCD, from Pseudomonas aeruginosa which utilizes 4-hydroxybenzoate as its sole carbon source, has a molecular This weight of 783,000and a composition of (n&Fe)s (7,8). enzyme has been crystallized and an X-ray diffraction study
938
Journal of Chemical Education
I 600 400
500
700
800
Wavelength (nm) Figure 1. Visible absorption spectra of protacatechuate 3.4diaxygenase complexes: E. native enzyme; ES, enzyme-substrate complex; ESO.. first interrnediate: ESOS', second intermediate.
troscopies have shown the native enzymes t o contain a highsoin ferric center (11312). EPR s~ectroscowv. .. which wrobes paramagnetic states of the metal center, reveals a signal at g = 4.3 fur these enzvmes. . . characteristic of a hiah - sum . ferric center in a rhombic environment (13). Mosshauer spectroscoov the nuclear states of S7Fe center and reveals .. orobes . dpectral parameters ior the enzymes consistent with a highswin ferric oxidation stace (10-12). The enzymes are must easily characterized by visible spectroscopy a h i c h shows a broad absorption band with a maximum near 460 nm and a molar extinction coefficient of 3000-4MH) per iron (Fig. 1) (1). Since ligand-field bands of high-spin ferric ions are both spin and orbitally forbidden and thus have small extinction coefficients (0.1-1 M-'cm-I), the visible absorption band has been assigned to a ligand-to-metal charge transfer transition. Considering the possible amino acid ligating groups, the two with hieh-lvina filled orbitals t o serve as donor orbitals are thiolate icysieine) and phenolate (tyrosine). Indeed.. earlv . studies favored the descriwtion of the dioxyaenases as iron-sulfur proteins (16) beca&e of EPR and visible swectral similarities to rubredoxin, a protein with a ferric cinter tetrahedrally coordinated t 4 , four thiolate groups. Resonance Itaman spectroscopy is an excellent technique for probing charge-transfer transitions. Raman spectroscopy provides vibrational information by measuring the ener-. gy of inelastically scattered light from a sample; the difference in the energies of the incident and scattered radiation corresponds to vibrational transitions. Normal Raman scattering is weak and thus usually impractical for dilute biologi-
cal samples. However, if the incident radiation approaches the energy of an allowed electronic transition, such as a charge transfer hand. the vibrational modes that are vibroni.--.~--~ ~~, cally coupled to the electronic transition will be enhanced by factors of 102- 106. deoendiwe on the molar extinction coeffi& dioxygenases are probed cient of the transitioh. ~ h the with radiation within their absorption envelopes, resonanceenhanced Raman features a t -1170, 1270, 1500, and 1600 cm-' are ohserved (17-21). These have been identified as characteristic vihrations of tyrosinate, thus the assignment of the visible band as tvrosinate-to-Fe(II1) charge transfer. Based on a normal mode analysis (22), the vibrations have vc.0, and two vc.c's, respecbeen assigned as principally 6c.~, tively. In contrast, iron-sulfur proteins exhihit Raman features near 370 and 800 cm-', corresponding to U F . . ~ and uc.s, resoectivelv ~" (23). , . The dioxygenase Raman spectrum is nicely reproduced by a model complex, Fe(salen)(OC6H4-4-CHd,the p-cresolate serving as the tyrosinate analog (Fig. 2) (4). The p-cresolate vihrations, readily identified by isotopic substitution experiments, correspond well to the assigned tyrosinate vihrations. In addition. the model comwlex exhibits features that arise from salen'vibrations, due-to the presence of a salen-toFe(II1) charge-transfer transition. The two transitions can be deconvoluted by an excitation profile study. Such a study involves obtaining Raman spectra at several wavelengths and determining ;he resonance enhancements of the various features re1atk.e to a standard which is not resonance enhanced. Features will exhibit greater or smaller enhancements depending on the extinction coefficient of their re~~
~
~
~~~~~~~
~~~
.~~~~
Figure 2. Resonance Ramsn specbum of Fe(sa1en) (OCBH4-4-CHa)in C O K N with 647.1 nrn excitation. Features marked S are mlvsnt vibrations.
Figure 3. Excitation profile of Fe(sa1en) (OCeH,4-CH,)
vibrations.
Figure 4. Resonance Raman spectra of PCD-inhlbltor complexes (514.5 nrn) excitation).
Volume 62 Number 11 November 1985
939
spective transitions a t a particular wavelength and the intensities are plotted as a function of wavelength. Figure 3 shows the excitation profile of Fe(salen)(OC8H4-4-CH3)and illustrates the relative energies of the salen-to-Fe(II1) and the p-cresolate-to-Fe(II1) charge-transfer transitions. Resonance Raman studies on other dioxveenase comolexes provide additional insights into the coo;&nation chemistry of the iron center (19,24,25). Phenols, for example, are good inhibitors of the dioxygenases. Phenol binding results in a blue shift of the visible spectrum, indicating some structural alteration of the active site. Raman studies (Fig. 4) show that the inhibitor phenol coordinates to the iron center without displacing the endogenous tyrosinates. The coordination of the inhibitor is indicated by the presence of additional Raman features; these vary in energy as different phenols are introduced and are shifted by isotopic substitution. For example, the features a t 1276 and 1597 cm-I shift upon ring deuteration of the 4-hydroxybenzoate in the PCD4-hvdroxvbenzoate comolex and are thus assiened to the inhibitori24). ~ o m ~ a r a t ' i studies ve on the PCD:~-chloro-4hydroxybenzoate complex and the PCD-4-hydroxybenzoate complex indicate that two tyrosines are involved in coordinating the iron center (19.24). The Raman soectrum of the former complex exhibits a vi.0 for the tyrosines which is approximately twice as intense as the uc.0 of the inhibitor phenol. Similarly, the l'C1)-4-hydroxybenzoatp romplex exhibits three nearly equally intense uro'a, only one of which nris~sfrumthe inhibitor.'rhusthe iron cencer is coordinated mtwoendogenouq tyrusinatesand hasasiteavailable for the coordination of exonenous lieands. The remainine lieands on the iron are proposed to ge histidines (26) a i d kater(or hvdroxide). (27) . . to eive rise to a five- or six-coordinate (28) . . metal center. Substrate binding to the dioxveenases in the absence of oxygen results in th;! development of absorption in the long wavelength region - (Fig. - 1) (29. 30). The oersistence of the \,isible spectrum suggests that the iron center is not reduced in this complex, as had been suggested earlier. Reduction of the iron center would have resulted in the bleaching the charge transfer band. EPR and Mossbauer studies corroborate t h e ferric nature of the iron in the enzyme-substrate (ES) complex (10-12). Raman studies on the ES complexes with 647.1 nm excitation (21) reveal new resonance-enhanced features a t -1260, 1320, and 1470 cm-I. These are due onlv to the substrate. as indicated bv" soectral shifts . when ring-deuterated substrate is used. No tyrosinatevibrations are observed with 647.1 nm excitation in the ES complexes in contrast to parallel experiments on the native dioxygenases wherein they are observed (19). Based on this observation, one may suspect that catechol has coordinated to the iron center and displaced the tvrosines. However, when the excitation wavelength is shifted to 514.5 nm, both catecholate and tyrosinate vibrations are observed. Therefore, catecholate hinding has the effect of shifting the tyrosinateto-Fe(II1) charge-transfer transition to higher energies. A perusal of the various dioxygenase complexes reveals a spectrum of colors (31). The native enzymes are burgundy, phenol complexes are yellow-brown, carboxylate complexes are purple, thiophenol complexes are orange-brown, cyanide complexes are green, and substrate complexes are purplish gray. In all cases, the tyrosinate-to-Fe(II1) charge-transfer hand oersists. as indicated bv the presence of tvrosinate vibrations in the resonance ~ a k a spectra n of the cbmplexes (19. 24. 25). These ohservations indicate that the chargetransfer transition is sensitive to the nature of the otier ligands in the iron complex and that the charge-transfer hand may he used to deduce the identity of an unknown ligand. A systematic study on a series of Fe(salen)X has investigated the factors that affect the energy of the phenolate-toFe(II1) charge-transfer hand (4). From a combination of ohservations from visible spectroscopy, paramagnetic NMR
Figure 5. Visible absorption speetra of Fe(m1en)X complexes illustrating the shifts of the salen-to-Fe(ll1) chargewander band.
-
I
940
Journal of Chemical Education
spectroscopy, Raman excitation profiles, and cyclic voltammetry measurements, i t is concluded that the ligand field strength of X affects the energy of the charge transfer band; the better ligand X is, the higher the energy of the salen-toFe(II1) charge transfer transition (Fig. 5). Based on these studies, a spectrochemical series has been developed. The phenolate-to-Fe(II1) charge-transfer transition shifts to hieher enerev as the series eoes from left to right. This series wzl he usef;l in deducing ;he nature of the &nsient intermediates in the dioxveenase reaction. The reaction oithe dioxygenase EScomplexes withdioxynen has been investigated with sto~ped-flowkinetics (32, 33). In most cases, two transient interkediates are observed and their rates of formation and decomposition can be calculated. From these rate constants, the bisible spectra of the intermediates can he constructed; Figure 1shows the spectra of the intermediates in the reaction-of the PCD-protocatechuate complex with 02.The sequence for the reaction is E, ES, ES02, and ESOz*. The native enzyme is obtained upon the decomposition of the second intermediate. Note that visible absorotion remains in these intermediates. thoueh the hand shijts from one species to the next. This kdicaies the persistence of the tyrosinate-to-Fe(II1) charge-transfer hand as the exogenous ligand is transformed from substrate to product. The iron center would appear to be in the ferric .. staie in these intermediates. The observation of the ferric oxidation state in the native enzyme, the ES complex, and all the transient intermediates suggest the possibility that the iron does not undergo reduction duringthe catalytic cycle. Indeed the presence of tyrosines in the metal coordination environment serves to stabilize the ferricoxidation state (4) and has aorofoundeffect on the mechanism of action for these enzymes. Earlv mechanistic oostulates reauired the bindine of dioxygen to a ferrous center to serve as the oxygen activation step. Since the native enzyme was known to have a ferric center, it was proposed that substrate binding resulted in the
-
M
o p o
Figure 6. Proposed mechanism fw the inhadiol cleaving catechol dioxygenases.
reduction of the ferric center followed by oxygen hinding and product formation. However, the spectroscopic data accumulated to date argues against such a mechanism, since the ES complexes are cfearly ferric complexes. This data has forced a reevaluation of the proposed mechanism and has resulted in a different proposed mechanism, one which postulates that the ferric center activates the suhstrate for reaction with catechol(3). This mechanism is illustrated in Figure 6. In the proposed mechanism, the substrate catechol is postulated to coordinate to the iron through only one oxygen. The coordinated substrate then acts as the reductant for oxygen, yielding as the suhstrate-oxygen adduct a peroxy species. The decom~ositionof the Deroxv intermediate is then iiicilitated by c&ordinationro the ironto yield an anhydride. Theopening of the anhydride iseffected bv rhe metalcoordinatedhydr~xide. Perhaps the novel aspect of this mechanism is the mode of catecholare hinding. Given the demonstrated affinity of ratechdate for trrrir ion i3.11,one would expect that the rntecholate would chelate to the ferric center. as has been ohserved for several iron catecholate complexes. However, it is this stabilization due to chelation that one would want to avoid in order to obtain a reactive species. Support for the monodentate mode of catecholate coordination has been obtained from a paramagnetic NMR study of the CTD euzyme-substrate com~lexes(35). Because of the piesence of the high spin ferric center, proton resonances of lizands coordinated to the iron will be shifted out of the diamagnetic region. The ferric center in effect acts as an internal paramagnetic shift reagent in the protein. Since high spin ferric ionhas a symmetric distrihution of d electrons, the paramagnetic shift observed will arise ~-~~ onlv from a contact mechanism...i.e...~~~~~~~ delocalization of- the unpaired spin density through bonds. Thus we may be able to distineuish between a monodentate and a chelated catecholate on the basis of NMR shift. For this one needs appropriate models. Such models have been svnthesized (31)and rheircrysral stnwturrs (36,37) are shown in Figure 7 . Fe(saIoph~mtHisanexamvleirfa monvdentatecatechol rornnlex. . . while ~ [ ~ e ( s a l e n ) c aisi ]the corresponding chelated catecholate complex. NMR spectra of the synthetic 4-methylcatecholate complexes are shown in Figure 8; 4-methvlcatechol has been chosen as the prohe zuhs-trare because the methyl-resonance iseniily identified. From an analysiiof the spectra, it isrleur
Figure 7. ORTEP plots ol the crystal structures of Fe(sa1oph)catHand K[Fe(salen)cat].
~
Figure 8. Paramagnetic 'H-NMR spectraof Fe(sa1en)complexesin acetone-4, Starred resonances are assigned to the CHJ group. that the different modes of catecholate hinding can be distinguished hy this technique (31). For monodentate catecholates, the methyl resonance appears a t -100 ppm downfield if the 0-1oxygen were coordinated to the iron and at -30 ppm upfield if the 0-2oxygen were coordinated. The same shifts are observed for methyl-substituted phenols. For chelated catecholates, the methyl resonance appears a t -50 Volume 62 Number
il
November 1965
941
ppm downfield.The NYR spectra of CTD ES complexesare shown in Fieure 9. C'omoarisonsof rhesoecrrum af the CTDcatechol complex with'that of the C~D-4-methylcatechol complex show the principal difference in the two spectra is a new feature in the latter a t 100 ppm downfield. This peak disao~earsin the 'H-soectrum when 4-(methvl-d&catechol . .. is used as suhstrnteanh is ohserved in rhe corresponding WN h l K wertrum. The data cleurls indicate that the substrate is coordinated to the ferric center solely through the 0 - 1 oxygen (35). The subsequent aspects of the mechanism have precedents in the hioorganic literature. The formation of the peroxy intermediate is analogous to the reaction of reduced flavin with oxygen to form the 4a-hydroperoxide (38), i.e.,
This peroxide species maybe the first intermediate obsewed in the stopped-flow studies (ES02 in Fig. 1). The blue shift of the charge transfer hand would be consistent with the coordination of the peroxide to the ferric ion, since the alkyl Deroxide would have a basicitv similar to that of nhenolate. The rearrangement of the peroxy intermediate to the desired oroduct can occur via the anhvdride as shown or via a dioxetane (39).The formation of the anhydride would be a result of a Crigee rearrangement (e.g., Baeyer-Villiger reaction), while the dioxetane is similar to intermediates in chemiluminescent reactions (40). For CTD, the intermediacy of the anhydride has been demonstrated by '80-labeling experiments (41). The remaining challenge for the bioinorganic chemist in this work is the duplication of this reaction in a small molecule. Proeress toward a model iron comolex caoable of catau lyzing this reaction has been encouraging. The initial reactivity studies on the Fe(sa1en)catecholate complexes proved disappointing (42). Though the monodentate complex was oxidized hv dioxveen and the chelated comolex was unreactive, as suggeste;lby the proposed mechanikns, the reactivity observed was simply a one-electron oxidation to a semiquinone complex. More recent results show that a ligand system like the tripodal NTA (nitrilotriacetic acid) is more effective. Weller and Weser have demonstrated t h a t Fe(NTA) in an organic solvent-buffer mixture catalyzes the oxidative cleavage of 3,5-di-tert-butylcatechol(DBC) in 80% yield based on substrate with as many as 100 turnovers (43). Crystals of [Fe(NTA)DBC]2-showa six-coordinate complex with the catecholate unsymmetrically chelated to the iron center (Fig. 10) (44). The Fe-0 (DBC) bond trans to N is 1.89 A,0.09 A shorter than the other Fe-O(DBC) hond; this is ascribed to the weakness of the Fe-N hond which is compensated by the stronger Fe-O(DBC) hond trans to it. This complex reacts with 0 2 in DMF over a period of four days to yield cleavage product in 80% yield. IROstudies show a very clean incorporation of a single oxygen label, strongly in support of an anhydride intermediate. The reactivity of the complex is proposed to result from the breaking of the weak942
Journal of Chemical Education
Figure 9. Paramagnetic 'H-NMR spectra ol cetschol 1,Mioxygenase ES complexes.
er Fe-O(DBC) hond generating the reactive monodentate catecholate complex.
The rate of the reaction is consistent with the high activation energy necessary to break the Fe-O(DBC) bond. Despite the slow rate of this reaction, it is clear that the enzyme chemistry can he mimicked in a synthetic system. It is hoped that this discussion has served to demonstrate the various approaches required to unravel the details of the structure and mechanism of a metallo-enzyme. Acknowledgments
This work was supported by the National Institutes of Health, the Alfred P. Sloan Foundation, and the University of Minnesota Graduate School Research Fund. The research described is the result of the accumulated efforts of R. H. Heistand, 11, R. B. Lauffer, R. J. Mayer, A. L. Roe, L. S. White, and J. W. Pyrz, to whom I am most grateful. Literature Cited (11 Que, L.,Jr.,Adu.Inorg.Bioehsm..5,167(19831. (21 Fei8t.C. T.. and Hegeman. G.D.,JBocteriol., l W , 1121 (1969); 0msten.L. N.,and Stanier,R. Y.. JBiol. Chem.,Z41,3776(19661. (31 Que, L..JI., Lipscomb, J. 0.. MU" \.",",.
(19) Fe1ton.R. H.,Cheung. L.D..Phillips,R.S..sndMsy,S.W.,Bioehem.Biophys.Re~. Comm., 86,844 (1978). (20) Buli,C.,Ballou. D.P.,andSslmecn,I., Riochem. Riophys:Rea. Comm.,81.836(1979). (21) Bue,L.,Jr.,Heistand,R.H., 11, J A m r r . Chem. Soc., 101,2219~1979). (22) Tomimateu,Y..Kint, S.,andSchenr, J.R.,Bioehamlalry. 15.4918(1976). (23) Yechandra,Y. K., Hare, J.,Moura,I., andSpiro,T. G.. J.Amer. Chem Sot, 105,6455
>.""",. !,a*">
(241 Bue, L.. Jr., aod Epstein, R. M., Bioehemiafry, 29,2545 (19811. (25) Bue, L., Jr., Heistand, R. H., 11, Mayer, R., and Roe,A. L.. Biochemistry, 19, 2588
Figure 10. ORTEP plot of t h e crystal structure of [Fe(NTA)OBC]2- (NTA, nitrilotriacetic acid: DBC. 3.5di-tert-butyl-catecholate dianion). (81 Kohlmiller, N. A,, and Howard, J. B., J.Rid. Cham.,254,7302 (1979); Kohlmiller. N. A . end H o d . J. B.. J. R i d Chem.. 254,7309 (1979). (91 Satyshur, K. A,, Rao, S. T., Liuscomb, J. D., and Wwd, J. M., J. B i d Chem., 255. ,m,s ,,Qnn, ~."""z. (10) Buc,L., Jr..Lipscomb,J. D..Zimmmnan.R., Munck,E.. OmreJohnson, N. R.,and Orma-Johnson, W. H., Rioehim. Biophya. A&, 452,320 (1976). (111 Whittaker. J. W.. Lipsmmh, J. D, Kent, T . A , and Munck, E., J. Bid. Chem., 259. 1166 119%) . I121 Kent.T. A., Munek. E., Whittaker. J. W..Lipammb. J. D.,Que,L.. Jt.,unpublished observations. (131 Ooeterhuia. W. T., Stnrcf. Bond. (Bedin).20. 59 (1974). (14) Nakazarva.T., Kojima, YYY Fuji~u~uw~u, H., N~saki,M.,H ~ ~ ~ i i h i , O . , oYoYd ~ Y Y Y ,J. T., B i d Chem.. 240. PC3224 (1965). I151 Fuiisawa, H., Uyeda, M., Kojima, Y.. Naski. M., and Haysishi, 0.. J. Biol. Chm., 247,4414 (19721.
.""."
~.
(26) Felton.R.H.,Ba.rm, W. L.,May.S. W..Soweli.A.L.,Goel,S.,Bunker,G.,Stem,E. A. J. Amsr Chem. SOE..22.6132 (1982). (271 Whittaker. J. W.. and Lipsmmb, J. D.. J. Biol. Chm., 259,4487 (19%). (28) Roe. A. L., Schnoider. D. J., Mavcr, R., P y n , J . W., Widom, J., and Bus, L., Jr.. J. Amar. Cham.Soc., 196 1676 (19%). (29) Kojima. Y., Fujisawa, H.. Nakazewa. A , Naksrawa.T., Ksncteuna. F., Taniuchi, H., Nazaki. M.. end Hawishi, 0..J. Rid. Chem.. 242,3270 (1967). (30) Fujisaw. H.. and Hwaishi, 0.. J. Bid. Chrm.. 243,2673 (1968). (31) Heiatmd. R. H., 11, Lauiior. R. B., Fikrig, E.. and que. L.. Jr., J. Amsr. Chem. Sac.. 104.2789 ..(19RK ,-. .,. . (32) Bull, c., Ballou, D. P. and Otsuka. S., J. Bid. Chom., 256,12681 (1981). (331 Walsh, T. A,, Ballou, D. P., M w r , R., and Que, L.. Jr., J. B i d Chem., 258, 14422 (19831. (341 Raymond,K. N..Isied.S. S.,Bro.n,L.D., FmnFrnek,F. R..~ndNibbbt,J.H.,J, Amilil. Cham Soe., 98,1767 (1975). (35) Laufier. R. B., and Bue, L., Jr., J. Arne,. Chem. Soc., 104,7324 (1982). (36) Heistand, R. H., 11, Roe, A. L., andQue, L., Jr., 1"-q. Chem., 21.676 (1962). (371 Lauiier, R. B., Hei8tand.R. H.,II,sndQue,L., Jr.,lnor~.Chem.22,50(1983). (381 Walsh. C.T.,"Enrymstie Resetion Meehanisma;.Freeman, San Francisco, 1971,pp. 390,413. (391 Hamilton.G.A.,in"MolecularMechaniamsofO~genAbivation"(Edifor:Hsyaishi, 0.1. Academic Pleas. New York. 1974. p. 405. (40) Hiatt, R.. in "Organic Peroxides,). Vol. 11, (Editor: Swern. D l . Wilay Interscience. New York. 197l.p. 1. (41) Mayor, R. J., and Buc, L., Jr., J. R i d Chem., 259,13056 (19%). (421 Laufier, R. B., Haiaand. R. H.. 11, and Bue. L., Jr., J. Amer. Chem. Soe., 103, 3947 (19811. (431 Weliar, M. G., and Wncr, U.,J. Amer Chem. Soc., 104,3752 (1982). (44) While, L.S., Nilsson, P. V.. Pigndet,L. , - . H 8312 (19841.
Volume 62
Number 11
November 1985
943
