EPR and MCD Studies of Oxomolybdenum Centers in Sulfite Oxidase

Chapter 21

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EPR and MCD Studies of Oxomolybdenum Centers in Sulfite Oxidase and Related Model Compounds John H. Enemark Department of Chemistry, University of Arizona, Tucson, AZ 85721

The minimal dithiolene coordination of the molybdenum center in sulfite oxidase (SO) has been modeled by LMoO(tdt), where L is hydrotris(3,5dimethyl-l-pyrazolyl)borate and tdt is toluene dithiolate. The MCD spectrum shows a low-energy (~9,000 cm ) band that is assigned to a Sπ->ΜΟ 4d charge transfer transition, and which is consistent with the occurrence of g values > 2 in the EPR spectrum. Electron spin echo envelope modulation (ESEEM) spectroscopy on the low pH and phosphate forms of SO unambiguously demonstrates that they contain Mo-OH and M o - O P O units, respectively. The crystal structure of chicken liver SO shows five-coordinate square-pyramidal geometry about the Mo atom with an axial oxo ligand; the equatorial donors are two S atoms from the dithiolene fragment of molybdopterin, a S atom from a cysteinyl residue, and an OH (or H O) ligand. -1

i

3

2

Sulfite oxidase (SO), an essential enzyme for sulfur metabolism in animals (7), is a member of a diverse group of enzymes that contain a mononuclear Mo center and which catalyze two-electron oxidation or reduction reactions by what is formally an oxygen atom transfer reaction (2-6). Specifically, SO catalyzes the oxidation of sulfite to sulfate with the concomitant reduction of two equivalents of ferricytochrome c (equation 1): 2

ffl

S0 " + 2(cytc ) + H 0 ^ S 0 3

2

r 4

u

+ 2(cytc ) + 2H

+

(1)

Hille (6) has subdivided this group of molybdoenzymes into three families based upon their reactivity and spectroscopic characteristics. Crystal structures are now available for at least one member of each family: aldehyde oxidoreductase (AOR) from the xanthine oxidase family (7); DMSO reductase (8-11) and formate dehydrogenase (72) from the DMSO reductase family; chicken liver sulfite oxidase (vide infra, 13,14) from the sulfite oxidase family. A key structural feature of each family is the coordination of the Mo atom

360

©1998 American Chemical Society In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


361

by the dithiolene fragment of a novel pyranopterin ligand (structure 1). In some bacterial enzymes the phosphate group is replaced by a dinucleotide, and for enzymes in the DMSO reductase family there are two pterins per Mo atom. Dithiolene coordination of the Mo atom by a substituted pterin was originally proposed by Johnson and Rajagopalan (75). The structure of the novel pyranopterin ligand of 1 was first determined in the tungstencontaining AOR from the hyperthermophilic bacterium Pyrococcus furiousus, which contains two pyranopterins per tungsten atom (16). Sulfite oxidase was first isolated, purified and shown to contain molybdenum in 1971 (77), but its X-ray structure has only recently been determined (13,14). The electronic spectrum of the enzyme is dominated by the absorptions of the £-type heme domain of the protein, but the Mo center has been probed directly by EXAFS at the Mo Kedge (18-20) and by EPR spectroscopy (21-25). These spectroscopic investigations of the Mo center of SO as well as recent studies of mutants of the enzyme (26) led to a proposed structure for the oxidized resting enzyme in which a dioxo-Mo(VI) center is coordinated by the dithiolene group of the novel pyranopterin (1) as well as a sulfur atom from a cysteinyl residue that is invariant in all SO enzymes (6,20). During turnover, SO cycles among the Mo(iV,V,VI) oxidation states; reoxidation of the enzyme by the physiological oxidant cyctochrome c is coupled to intramolecular electron transfer from the Mo center to the &-type heme of SO. The paramagnetic Mo(V) state has been extensively studied by CW-EPR, and three spectroscopically distinct forms of SO have been identified: one form is obtained at high pH (9-9.5) in low concentrations of chloride or phosphate; the other two forms are observed at low pH (6.5-7) (21-24). The particular low pH form which is observed depends upon whether phosphate is present in the buffer (22). In the absence of phosphate the CW-EPR spectrum of SO gives the so-called "low pH signal" which is characterized by a hyperfine interaction with a strongly coupled exchangeable proton (assigned to a Mo-OH group) and g > 2 (22,25). For Mo(V), simple ligand field theory predicts that all g < 2 (27); the large g-value for SO and other molybdoenzymes is characteristic of sulfur coordination to Mo(V), as occurs in structure 1 (28). 7

t

Model Oxo-Mo(V) Compounds In order to investigate the effect of a single coordinated dithiolene on the EPR parameters of an oxo-Mo(V) center, compound 2 (Figure 1) was prepared as a minimum structural model for 1 and the Mo center of SO (25). Comparison of the EPR parameters of 2 and of the different forms of SO (Table I) shows that this simple model closely mimics gj and g observed for SO at low pH in the absence of phosphate, but that g for the enzyme is larger than for 2. These EPR data suggest that SO has at least three sulfur atoms coordinated to the Mo atom, as has been proposed from EXAFS (18-20) and mutation studies (26). Single crystal EPR spectra of 2 diluted in a diamagnetic host lattice of LMo(NO)(bdt) are most consistent with the orientation of the g-tensor shown in Figure 2 (29,30). Compound 2 has approximate C symmetry; the largest g component lies in the MoS plane approximately perpendicular to the mirror plane, and the second largest component is approximately parallel to the Mo=0 bond. These EPR studies of 2 confirm what has long been known; sulfur ligation of oxo-Mo(V) centers increases the g-values 2

3

s

2

In Spectroscopic Methods in Bioinorganic Chemistry; Solomon, E., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


Figure 1. Perspective view of the structure of 2; Mo-Sl = 2.368(2), Mo-S2 = 2.379(2); Mo-O = 1.678(4) Â (reproduced with permission from ref. 33. Copyright 1996 American Chemical Society).

Figure 2. Orientation of the g-tensor of 2 relative to the molecularframeof LMo(NO)(tdt), the diamagnetic host for 2. The largest component (g ) lies approximately in the MoS plane and is perpendicular to the mirror plane of the molecule; g is nearly parallel to the Mo=0 bond (reproduced with permission from ref. 29 ). 7

2

2


363 Table I EPR Data for Sulfite Oxidase and Selected Mo(V) Compounds


Species

gj

g

g

2

3

Reference

sulfite oxidase (low pH, no phosphate)

2.007

1.974

1.968

1.983

34

sulfite oxidase (low pH, phosphate)

1.962

1.970

1.993

1.975

31

sulfite oxidase (high pH, no phosphate)

1.990

1.966

1.954

1.970

34

LMoO(bdt)2

2.004

1.972

1.934

1.971

34

LMoO(cat)

1.969

1.969

1.919

1.952

38

9597

(and decreases the A( Mo) hyperfine parameters) (28). However, the EPR results alone do not provide insight concerning the features of the electronic structure of 2 that lead to large g-values. Magnetic circular dichroism (MCD) spectroscopy is an extremely powerful technique for investigating the electronic structures of metal centers (32). Figure 3 shows the electronic absorption spectra for two isostructural oxo-Mo(V) complexes that differ in the number of sulfur atoms coordinated to the Mo atom; LMoO(cat) has only oxygen and nitrogen donor atoms, whereas LMoO(tdt) possesses a dithiolene ligand as in 2. Even cursory inspection of Figure 3 shows that the coordination of a dithiolene ligand leads to a low energy absorption band (ca. 9,000 cm") that is absent in the analogous catecholate complex. In addition, LMoO(tdt) exhibits a much richer MCD spectrum than LMoO(cat). These spectral differences have been rationalized by the molecular orbital energy level diagram derived from Fenske-Hall calculations (Figure 4) (33,34). These calculations indicate that the low energy band at ca. 9,000 cm" in LMoO(tdt) arises from charge transfer transitions from filled sulfur ρπ orbitals to the half-filled Mo 4d orbital that lies in between the ligands in the xy plane. This charge transfer band is relatively weak (e ~ 1000 M cm ) because the orbitals involved in the transition are nearly orthogonal to one another. However, these low-lying charge transfer states and spin-orbit coupling of the sulfur atoms (ca. 370 cm ) can account for one of the g values being greater than the free electron value (g = 2.0023) according to equation 2 (34-37). Each summation in equation 2 is over all appropriate excited states; ζ is the single-electron spin-orbit coupling constant for an electron in a metal d orbital; F and G are terms that depend upon the composition of the molecular orbitals in the ground and excited states and ligand spin-orbit coupling contributions; AE . is the d-d transition energy and A E is the energy associated with a single-electron excitation from a filled molecular orbital of mainly ligand character to the half-filled metal d orbital of the ground state. Comparable contributions from the two terms of opposite sign in equation 2 could result in gj > 2 (Table I). For LMoO(cat) all g < 2 because the second term in equation 2 is neglible due to the large value of A E and the small spin-orbit coupling constant for oxygen (38). 1

1

_1

_1

1

f

e

Μο

d

d

CT

t

CT



364

Figure 3. Electronic absorption (dark line) and MCD (light line) spectra for a) LMoO(cat) and b) LMoO(tdt) (reproduced with permission from ref. 32. Copyright 1994 American Chemical Society).



365

-6 H

-9-1 Figure 4. Partial molecular orbital energy level diagram for LMoO(tdt) and related compounds showing the filled sulfur ρπ orbitals that can be involved in low energy charge transfer bands to the half-filled Mo 4d orbital in structures such as 2 (adapted from ref. 32 and reproduced with permission from ref. 33. Copyright 1996 American Chemical Society).


366

g, = 2.0023 - Σ

'C M /

\

d-d)

+ yί ' • A {Mer)

(2)


ESEEM Studies of Sulfite Oxidase The well-known proton hyperfine splitting observed for SO at low pH in the absence of phosphate has been assigned to an Mo-OH group (22). This proton hyperfine splitting is not observed in phosphate buffer (22), and Bray and coworkers obtained evidence for coordinated phosphate from careful difference CW-EPR measurements between 0labelled phosphate and ordinary phosphate (23). Electron spin echo envelope modulation (ESEEM) is a powerful technique for detecting and quantitating weak hyperfine and quadrupole interactions (39,40). This technique has been used here to probe in detail the Mo(V) center of SO at low pH in the presence and absence of phosphate. The ESEEM spectra of SO in D 0 at low pH are shown in Figure 5. In the absence of phosphate (top trace) several deuteron frequencies are observed, including a strong peak near 8 MHz assigned to 2v . A detailed analysis of this 2 v peak as a function of field using four-pulse ESEEM methods shows a deuterium quadrupole splitting of0.28-0.30 MHz, which clearly indicates the presence of an Mo-OD group and not an Mo-OD group (Raitsimring, A.; Enemark, J. H.; Pacheco, A. to be published). In phosphate buffer (bottom trace) the strong peak near 8 MHz disappears and a new peak appears near 20 MHz due to 2v , thereby providing unambiguous direct evidence for coordinated phosphate under these conditions. Multifrequency ESEEM investigations of the Mo(V) center of SO in phosphate buffer suggest that the coordinated phosphate group is monodentate and that it adopts a distribution of orientations (structure 3) (41). Combining the ESEEM results on SO in phosphate buffer with other spectroscopic, chemical, and mutation (26) data for the enzyme lead to the structural proposal for catalysis and inhibition by phosphate shown in Scheme 1. Initially the reduced form of SO is generated by direct oxygen atom transfer to sulfite, as proposed by Brody and Hille (42). Displacement of the product, sulfate, by water is a necessary step in the regeneration of the starting Mo(VI) species. High phosphate concentrations lead to the formation of the phosphate-inhibited form of the enzyme either by direct displacement of sulfate or by funneling off the hydroxo species (41). 17

2

D

D

2

P

Structure of Sulfite Oxidase SO is an a dimer with a total mass of about 110 kDa, and each subunit contains a 6-type heme domain and one molybdenum center (7 7). Preparation of diffraction quality crystals has proved to be frustratingly difficult over the years because the protein tends to form very thin plates. The preparation of ultra pure enzyme in our laboratory has finally led to crystals that diffract to 1.9 Â with the use of synchrotron radiation and has enabled the structure of chicken liver SO to be determined very recently (13,14). The approximately square pyramidal coordination geometry about the molybdenum atom is shown in Figure 6. 2


0.08

-

0.07

-

0.06

"

ESEEM spectra of sulfite oxidase

W

0.05

Lu

1

0.04

-

FFT


367

0.03

-

0.02

-

0.01

-

0.00

^

'S.

20

30

frequency/ MHz

Figure 5. Two pulse ESEEM spectra of sulfite oxidase in D 0 at pH* = 6.5. The top trace shows the spectrum in the absence of phosphate; the strong peak near 8 MHz is due to 2v of the Mo-OD group. The bottom trace shows the spectrum in phosphate buffer; the Mo-OD peak is absent and a new peak apppears near 20 MHz due to 2v fromthe coordinated phosphate group. 2

D

P


368


J

1

F 6 " [•-

Lr±}-

ο*

S-CH

2

s^llv^-so. S-CH2

so5 +Ft H,PCT

4

H£> Q

I

Fe"

[-

s

x

\

Fe

/

S-CH -< 2

1

]

ίΐ \

b

s

x

Ο

Il y Mo

^PO H 3

\

/

S-CH -< 2

inhibited form catalytic cycle Scheme 1.

Figure 6. Coordination environment about the Mo atom in chicken liver SO; Mo-O(l) = 1.7, Mo-0(2) = 2.3, Mo-Scys = 2.5, Mo-Sl = 2.3, Mo - S2 = 2.4 Â.



369 A terminal oxo group (Mo=0, 1.7Â) occupies the axial position, and the equatorial positions are occupied by sulfur atoms from the dithiolene unit of the pyranopterin (1) and from a coordinated cysteinyl residue that is invariant among all sulfite oxidases and nitrate reductases (6). The remaining equatorial position is occupied by an oxygen atom (Mo-O, 2.3 Â) that is most reasonably described as a coordinated water or hydroxide. The position trans to the Mo=0 group is blocked by protein residues and not accessible to solvent or anions. The observation of two very different M o - 0 distances is surprising because E X A F S studies of the oxidized resting form of the enzyme have indicated a dioxo-Mo(VI) core with an Mo=0 distance of 1.71 Â (18-20). The strikingly inequivalent distances observed in the crystal structure are suggestive of a monooxo-Mo(IV) center. However, the actual oxidation state of the Mo atom in the crystals is unknown at this point, and there is no direct evidence for the active oxidized enzyme becoming reduced either during crystallization or data collection. The proposed chemical mechanisms of SO (43), including that shown in Scheme 1, involve intramolecular electron transfer from the Mo center to the £-type heme center during turnover. The Mo—Fe distance in SO (32 Â) is surprisingly long considering the rates observed for turnover and intramolecular electron transfer. Moreover, the pterin ring, which has been invoked as part of the electron transfer pathway in other molybdenumcontaining enzymes (7-12) is not even oriented in the direction of the Z?-type heme in SO (13,14). Summary Spectroscopic studies of molybdenum-containing enzymes and molybdenum model compounds have been an active area of research since the first observation of a molybdenum EPR signal from xanthine oxidase in 1959 (44). However, only in the past two years have crystal structures of these enzymes begun to become available (7-14). The very recent determination of the structure of chicken liver SO (13,14) raises questions about the actual oxidation state of the Mo atom in the crystal, the mechanism of sulfite oxidation, and the pathway for intramolecular electron transfer over the 32 Â distance between the Mo and Fe centers. As is often the case, the solution of the crystal structure of SO has raised many new questions about the structure and function of its metal centers. It has been said that bioinorganic chemistry begins when the structure of the protein is known (Solomon, Ε. I. private communication). Following this dictum, 1997 marks the beginning of the bioinorganic chemistry of SO.

Ligand Abbreviations Ligand abbreviations: bdt = 1,2-benzenedithiolate; cat = catecholate; L = hydrotris(3,5dimethyl-l-pyrazolyl)borate; tdt = toluenedithiolate.


370 Acknowledgments


The contributions of coworkers P. Basu, M.D. Carducci, IK. Dhawan, A. Pacheco, E. P. Sullivan, Jr., W.A. Wehbi and B. L. Westcott to this research are deeply appreciated. I am indebted to C. Kisker, A. Raitsimring, D. C. Rees, H. Schindelin and Ε. I. Solomon for fruitful collaborations. Financial support from the National Institutes of Health (grant GM37773) and the Materials Characterization Program of the University of Arizona is gratefully acknowledged. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19.

Rajagopalan, K.V. Nutr. Rev. 1987, 45, 321-328. MolybdenumEnzymes;Spiro, T. G., Ed.; John Wiley and Sons: N.Y., 1985. Molybdenum and Molybdenum-ContainingEnzymes;Coughlan, M.P., Ed.; Pergamon Press: Oxford, U.K., 1980. Enemark, J. H.; Young, C. G.; Adv.Inorg.Chem. 1993, 40, 1. Pilato, R. S.; Stiefel, Ε. I. In Inorganic Catalysis; Reedijk, J., Ed.; Marcel Dekker, Inc.: New Your, 1993;p131. Hille, R. Chem. Rev., 1966, 96, 2757-2816. Romão, M. J.; Archer, M.; Moura, I.; Moura, J. J. G.; LeGall, J.; Engh, R.; Schneider, M.; Hof, P.; Huber, R. Science 1995, 270, 1170-1176. Schindelin, H.; Kisker,C.;Hilton, J.; Rajagopalan, Κ. V.; Rees, D. C. Science, 1996, 272, 1615-1621. Schneider, F.; Löwe, J.; Huber, R.; Schindelin, H.; Kisker,C.;Knäblein, J. J. Mol.Biol.1996, 263, 53-59. Bailey, S.; McAlpine, A. S.; Duke, Ε. M. H.; Benson, N.; McEwan, A. G. Acta Cryst. 1996, 52D, 194-196. Bailey, S.; McAlpine, A. S.; McEwan, A. G.; Shaw, A. L.J.Biol.Inorg.Chem. 1997, in press. Boyington, J.C.;Gladyshev, V. N.; Khangulov, S. V.; Stadtman, T.C.;Sun, P. D. Science 1997, 275, 1305-1308. Kisker,C.;Schindelin, H.; Rees, D.C.;Pacheco, Α.; Enemark, J. H. Abstracts, Molybdenum Enzymes Meeting, University of Sussex, UK, April 12-15, 1997, P28. Kisker,C.;Schindelin, H.; Pacheco, Α.; Wehbi, W. Α.; Garrett, R. M.; Rajagopalan, Κ. V.; Enemark, J. H.; Rees, D. C., submitted for publication. Johnson, J. L.; Rajagopalan, Κ. V. Proc.Natl.Acad. Sci. USA 1982, 79, 68566860. Chan, M. K.; Mukund, S.; Kletzin, Α.; Adams, M. W. W.; Rees, D. C. Science 1995, 267, 1463-1465. Cohen, H. J.; Fridovich, I.; Rajagopalan, Κ. V.J.Biol.Chem. 1971, 246, 374382. Cramer, S.P., Wahl, R., Rajagopalan, K.J.J.Am. Chem. Soc. 1981, 103, 7721. George, G.N.; Kipke, C.A.; Prince, R.C.; Sunde, R.A.; Enemark, J.H.; Cramer, S.P. Biochemistry 1989, 28, 5075.


371 20. 21. 22. 23. 24.


25. 26. 27. 28.

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

George, G. Ν.; Garrett, R. M.; Prince, R.C.;Rajagopalan, Κ. V. J. Am. Chem. Soc. 1996, 118, 8588-8592. Bray, R. C. Polyhedron 1986, 5, 591-595. Lamy, M. T.; Gutteridge, S.; Bray, R. C. Biochem. J. 1980, 185, 397-403. Gutteridge, S.; Lamy, M. T.; Bray, R. C. Biochem. J. 1980, 191, 285-288. Bray, R.C.;Gutteridge, S.; Lamy, M. T.; Wilkinson, T. Biochem. J. 1983, 211, 227-236. Dhawan, I.K.; Pacheco Α.; Enemark, J.H. J. Am. Chem. Soc. 1994, 116, 79117912. Garrett, R. M.; Rajagopalan, Κ. V. J.Biol.Chem. 1996, 271, 7387-7391. Mabbs, F. E.; Collison, D. Electron Paramagnetic Resonanceofd Transition Metal Compounds; Elsevier Science Publishers Β. V.: Amsterdam, 1992. W.E. Cleland, Jr., K.M. Barnhart, K. Yamanouchi, D. Collison, F.E. Mabbs, R.B. Ortega, and J.H. Enemark,Inorg.Chem. 1987, 26, 1017, and references therein. Westcott, B. L.; Dhawan,I.;Raitsimring, Α.; Enemark, J. H. Abstracts, 213th ACS National Meeting, San Francisco, CA, April 13-17, 1997, poster INOR504. Westcott, B. L., Jr. Ph.D. dissertation, University of Arizona, 1997. George, G.N.; Prince, R.C.; Kipke, C.A.; Sunde, R.A.; Enemark, J.H. Biochem. J. 1988, 256, 307. Pavel, E. G.; Solomon, E. I., this volume, and references therein. Carducci, M.D.; Brown,C.;Solomon, E.I.; Enemark, J.H.J.Am. Chem. Soc. 1994, 116, 11856-11868. Dhwan, I.K.; Enemark, J.H.Inorg.Chem. 1996, 35, 4873-4882. Glarum, S. H.; J. Chem. Phys. 1966, 41, 1125. Garner, C. D.; Hillier, I. H.; Mabbs, F. E.; Taylor,C.;Guest, M. F.;J.Chem. Soc., Dalton Trans. 1976, 2258. Garner, C. D.; Mabbs, F. E. J.Inorg.Nucl. Chem. 1979, 41, 1125. Basu, P.; Bruck, M.A.; Li, Z.; Dhawan I.K.; Enemark, J.H.Inorg.Chem. 1995, 34, 405-407. Dikanov, S.A.; Tsvetkov, Yu. D. Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy; CRC Press: Boca Raton, 1992; chapter 1. Willems, J-P.; Lee, H-I.; Burdi, D.; Doan, P.; Stubbee, J.; Hoffman, B. M., this volume, and references therein. Pacheco, Α.; Basu, P., Borbat, P., Raitsimring, A.M.; Enemark, J.H. Inorg. Chem. 1996, 35, 7001-7008. Brody, M. S.; Hille, R. Biochim. Biophys. Acta, 1995, 1253, 133-135. Sullivan, E.P., Jr.; Hazzard, J.T.; Tollin G.; Enemark, J.H. Biochemistry 1993, 32, 12465-12470, and references therein. Bray, R.C.;Malmström,B. G.; Vänngård, T. Biochem. J. 1966, 73, 193.


EPR and MCD Studies of Oxomolybdenum Centers in Sulfite Oxidase

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