11288
J. Phys. Chem. 1995,99, 11288-11291
Measurement of the Dipolar Interaction between an Oxomolybdenum Center and a Phosphorus Nucleus in Models for the Molybdenum Cofactor of Enzymes by Pulsed Electron Spin Resonance at S-Band V. V. Kurshev and L. Kevan* Department of Chemistry, University of Houston, Houston, Texas 77204
P. Basu and J. H. Enemark Department of Chemistry, University of Arizona, Tucson, Arizona 85721 Received: April 10, 1995; In Final Form: May 12, 1 9 9 9
Low-frequency pulsed electron spin echo modulation spectroscopy has been successfully applied to the study of the dipolar interaction between a molybdenum(V) center and a phosphorus nucleus in the model sixcoordinate oxomolybdenum(V) compounds, LMoO(cat-3-OPO(OPh)2) (I)and LMoO(cat4OPO(OPh)z) (II). L is the facially coordinating tridentate hydrotris(3,5-dimethyl- 1-pyrazolyl)borate ligand; cat-3-OPO(OPh)2 and cat-4-OPO(OPh)2 are 1,Zcatecholate ligands that contain pendant diphenylphosphate ester groups at the 3- and 4-position, respectively. The molybdenum-phosphorus interaction is detectable if the distance between molybdenum and phosphorus nuclei does not exceed 7-8 A. Experimental data obtained for I, the complex with the smaller molybdenum-to-phosphorus distance, cannot be explained by a single distance parameter, suggesting that the phosphate ester group of I adopts a distribution of rotational conformations in frozen toluene solution. The success of low-frequency pulsed electron spin echo modulation spectroscopy in detecting dipolar molybdenum-phosphorus interaction in 11 implies that this technique should be applicable to the study of the dipolar interaction between molybdenum(V) and phosphorus in the molybdenum cofactor (Moco) of enzymes.
Introduction Pulsed electron spin echo modulation spectroscopy at S-band (2-4 GHz) has some advantages over the more commonly used X-band frequency (9 GHz), since it gives a 10-fold increase in modulation amplitude.'-3 At the same time, the increased modulation amplitude reduces the electron spin echo signal amplitude4 due to the smaller Zeeman splitting in the lower magnetic field and due to the lower sensitivity of microwave field detectors at lower frequency. Thus, pulsed electron spin resonance spectroscopy at S-band demonstrates a clear advantage over that at X-band only for certain systems like E'-centers in y-irradiated silica and paramagnetic centers in coals. Molybdenum is an essential trace metal that is present in "oxo-type'' enzymes, such as xanthine oxidase, sulfite oxidase, and nitrate reductase?-9 These enzymes occur in diverse organisms and catalyze reactions involving a change in the number of oxygen atoms in the substrate. On the basis of a series of degradation studies, it has been proposed that these enzymes possess a common molybdenum cofactor (Mo-co) that contains a novel reduced pterin (molybdopterin) which coordinates the molybdenum atom by an enedithiolate side chain (Figure The side chain is terminated by a phosphate group. There is as yet no crystal structure for an "oxo-type" molybdenum enzyme, but very recently the structure of a related tungsten-containing enzyme, aldehyde ferredoxin oxidoreductase, has been reported.I2 This enzyme has two molybdopterin ligands per tungsten atom; the overall structure of each pterin fragment (Figure lb) is slightly different from that proposed by Rajagopalan and co-workers, but the structure confirms the presence of a pendant phosphate group with a metalphosphorus distance of about 7 A. For xanthine oxidase the @
Abstract published in Advance ACS Abstracts, June 15, 1995.
0022-365419512099- 1 1288$09.0010
a
b
Figure 1. (a) Proposed structure for molybdopterin and its coordination to molybdenum in Mo-co. (b) Structure found for the molybdopterin ligand in the tungsten-containing enzyme aldehyde ferredoxin oxi-
doreductase. terminal phosphate group has been detected by 31Pnuclear magnetic resonance (NMR).I3 The interaction of the magnetic 31Pnucleus (I = '/2, 100% abundant) with aparamagnetic Mo(V) center is a possible probe of the distance between the molybdenum and phosphorus atoms of Mo-co. In model complexes of Mo(V) containing a pendant phosphate ester (Figure 2) the molybdenum-phosphorus interaction was successfully measuredI4 by an analysis of the phosphorus NMR line broadening. Since electron spin resonance (ESR) is about lo00 times more sensitive than the NMR, and electron spin echo modulation (ESEM) is particularly sensitive to electronnuclear interactions, we have undertaken 31PESEM studies of the model complexes of Figure 2 using both X-band and S-band ESEM spectroscopy. While the weak modulation amplitude and interference from other magnetic nuclei (I4N, 'OB, 'B,'H) makes quantitative analysis at X-band practically impossible, numerical simulations at S-band demonstrate good sensitivity of the ESEM to the distance between the unpaired electron spin on molybdenum and the nearest phosphorus magnetic nucleus.
'
Experimental Section The preparation of complexes I and II (Figure 2) is described e1~ewhere.l~The samples contained 5 mM solutions of each 0 1995 American Chemical Society
Oxomolybdenum-Phosphorus Interactions
Complex I
J. Phys. Chem., Vol. 99, No. 28, 1995 11289
Complex
II
Figure 2. Molecular structure of the Mo(V) complexes I and 11. compound in toluene in 2 mm i.d. by 3 mm 0.d. Suprasil quartz tubes and were rapidly frozen to 77 K in liquid nitrogen. Three-pulse electron spin echo modulation at X-band was recorded at 4.2 K with a Bruker ESP 380 pulsed ESR spectrometer with a n/2-z-n/2-T-n/2 pulse sequence with a microwave pulse duration of 24 ns and z = 200 ns to maximize phosphorus modulation and minimize proton modulation with T as a variable interpulse time. Three-pulse ESEM experiments at S-band were obtained with a home-built pulsed ESR spectrometer with a n/2-z-n/2-T-n/2 pulse sequence with a microwave pulse duration of 30 ns and z = 240 ns.
Results Experimental ESEM spectra at X-band for complexes I and
II are given in Figure 3 together with their Fourier-transformed ESEM (FT-ESEM) spectra. Since the only structural difference between these two complexes is the location of the phosphoruscontaining group relative to Mo(V), we expected to see a difference in the FT-ESEM spectra near the 31P Zeeman frequency at about 6 MHz in a magnetic field of 3600 G. The dominant peak near 0.7 MHz in both spectra is presumably due to 14N. Its intensity is about 2 orders of magnitude stronger than the expected intensity near 6 MHz due to 31P,because I4N has a nuclear spin I = 1 while 31Phas a nuclear spin of l/2, and the distance from the unpaired electron to the nitrogen nuclei is less than that to the phosphorus. Such a big difference in the amplitudes near the phosphorus Zeeman frequency and the maximum of the spectrum makes the phosphorus FT-ESEM difficult to analyze, firstly because the typical analog-to-digital (A/D)converter range of 8 bits (as in the Bruker ESP-380) is not sufficient and secondly because small artifacts, like side-
bands on the main line, can overlap with the nearest weak line due to 31P and distort it. From the theory of ESEMI5 and numerical simulationsI6 it is known that the modulation amplitude due to distant magnetic nuclei is inversely proportional to the second power of the spectrometer operating frequency, while for nuclei close to the unpaired electron the modulation amplitude does not change with frequency. Therefore, an experiment at S-band can reduce the difference between strong and weak ESEM peaks, to better enable a quantitative analysis of the phosphorus modulation. Figure 4 shows experimental ESEM data at S-band and the corresponding FTESEM for Mo(V) complexes I and 11. The FT-ESEM spectra show clear differences in the range 1.2-2.0 MHz.
Analysis and Discussion Quantitative simulations of the phosphorus modulation require a complex nearly identical to I and 11, with an FT-ESEM spectrum showing the same interaction with all magnetic nuclei except 31P. For this purpose we used the two model complexes in which either HI7 or OH functional groups replaced the phosphate ester group in complex I (see Figure\2). Unfortunately, these attempts were not successful because the FT-ESEM spectra for both of these molybdenum complexes were only slightly different from those in Figure 4, and some of the difference was outside the 31P region. This difference was possibly caused by changes in the unpaired electron density distribution and concomitant changes in the quadrupole and hyperfine interaction parameters that were induced by changing the substituents on the catecholate ligand system. To obtain a FT-ESEM spectrum without a 31Pinteraction, we used the following procedure. The 31Pnucleus of complex I1 is 7.3 A from the unpaired electron spin, and this distance can vary only slightly ( f 0 . 4 A) due to possible rotation of the phosphorus-containing group around the C-0 bond. Since an experimental ESEM can be described as a product of modulations due to all nuclei interacting with the unpaired electron spin, the experimental ESEM of complex I1 was divided by a simulated ESEM due to one 31Pat a distance of 7.3 A with an isotropic hyperfine interaction constant equal to zero. Subsequent Fourier transformation of the resulting ESEM gives a FTESEM for all interacting nuclei in the complex except 31P,as shown in Figure 5 together with the FT-ESEM from complex
$0.6
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Complex 1 Fr of ESEM
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Figure 3. Experimental three-pulse ESEM for Mo(V) complexes I and I1 at X-band and their Fourier-transformed (FT) ESEM spectra.
Kurshev et al.
11290 J. Phys. Chem., Vol. 99,No. 28, I995 1.or
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. A
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9 0.2
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Figure 6. Experimental FT-ESEM for Mo(V) complex I at S-band (solid line) and a simulation for Mo-P distances of 0.45 nm (dashed line) and 0.62 nm (dotted line).
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Figure 4. Experimental (a) three-pulse ESEM for Mo(V) complexes I and I1 at S-band and (b) their Fourier-transformed (FT)ESEM spectra.
MHz
Figure 7. Experimental ESEM for Mo(V) complex I at S-band and the simulated FT-ESEM spectrum corresponding to the distribution of Mo-P distances due to rotation of the phosphrous-containinggroup around the C-0 bond.
MHz
Figure 5. Experimental FT-ESEM for Mo(V) complex I1 at S-band (solid line) and the spectrum obtained after elimination of the 31P contribution to the spectrum (dashed line) according to the procedure described in the text.
11. The small difference between the two spectra in Figure 5 indicates that 7-8 A is approximately the maximum distance for which the interaction of the unpaired electron with a magnetic nuclear spin of '12 can be detected at S-band. Starting from the FT-ESEM without 31P modulation obtained according to the above procedure, one can simulate an FTESEM for a complex with a 31Pnucleus at any distance from the unpaired electron by multiplying the ESEM without an electron-phosphorus dipole interaction by a simulated ESEM due to one phosphorus nucleus at a given distance and subsequent Fourier transformation. Figure 6 shows the spectra obtained by this procedure for unpaired electron-phosphorus
distances of 4.1 and 6.3 A, the minimum and maximum possible distances between 31Pand the unpaired electron in complex I, allowing for rotation around the C-0 bond. Other simulations with one 31Pat one fixed distance from the unpaired electron spin were not significantly better than those in Figure 6. A better fit is an averaged sum of 10 31P ESEM corresponding to various values of the 0 - P azimuthal angle (0, n15, 2 ~ 1 5 ..., , 9nb) of rotation around the C-0 bond (Figure 7). This indicates that complex I does not have a single rotational conformation of the pendant phosphate ester p u p , but instead has a distribution of orientations.
Conclusions Experimental results and numerical simulations of ESEM at S-band show that one spin '12 magnetic nucleus can be detected at a distance of 7-8 A, which is a greater distance than detectable from ESEM at X-band. Lower-frequency pulsed ESR spectroscopy can be especially informative for the analysis of weak interactions of the unpaired electron with distant magnetic nuclei even when their IT-ESEM spectra are complicated by interactions with other nuclei. Computer simulations of 31P ESEM indicate that the phosphate ester group of I has a distribution of rotational orientations in frozen solution in toluene. Finally, these preliminary results on model compounds I and 11hold promise for using low-frequency pulsed electron spin resonance to study the molybdenum-phosphorus interaction in Mo-co in the Mo(V) state(s) of enzymes. There is not
Oxomolybdenum-Phosphorus Interactions yet a crystal structure available for any enzyme that contains Mo-co. However, the structure of the tungsten-containing aldehyde oxidase from Desulfovibrio gigas has recently been reported. The distance from the tungsten atom to a phosphorus on the side chain of the pterin is about 7.5 A,’8 within the distance limits of the present model study.
Acknowledgment. This research was supported by a National Science Foundation grant to L.K.and by a National Institute of Health grant (GM-37773) to J.E. The authors thank Amold Raitsimring for stimulating discussions. References and Notes (1) Clarkson, R. B.; Timken, M. D.; Brown, D. R.; Crookham, H. C.; Belford, R. L. Chem. Phys. Lett. 1989, 163, 277. (2) Hankiewicz, J. H.; Stenland, C.; Kevan, L. Rev. Sci. Instrum. 1993, 64, 2850. (3) Kurshev, V. V.; Buckmaster, H. A.; Tykarski, L. J. Chem. Phys. 1994, 101, 10338. (4) Romanelli, M.; Kurshev, V. V.; Kevan, L. Appl. Magn. Reson. 1994, 6, 427. ( 5 ) (a) Rajagopalan, K. V. In Biochemistry of the Essential Ultratrace Elements; Frieden, E., Ed.; Plenum Press: New York, 1984; p 149. (b) Rajagopalan, K. V. Nutr. Rev. 1987 45,321. (c) Burgmayer, S. J. N.; Stiefel, E. I. J . Chem. Educ. 1985, 62, 943.
J. Phys. Chem., Vol. 99, No. 28, 1995 11291 (6) Johnson, J. L. In Molybdenum and Molybdenum Containing Enzymes; Coughlan, M. P., Ed.; Pergamon Press: New York, 1980; p 345. (7) Garner, C. D.; Bristow, S. In Molybdenum Enzymes; Spiro, T. G., Ed.; Wiley: New York, 1985; Chapter 7. (8) Pilato, R. S.; Stiefel, E. I. In Bioinorganic Catalysis; Reedijk, J., Ed.; Mace1 Dekker: New York, 1993; pp 131-188. (9) Enemark, J. H.; Young, C. G. Adv. Inorg. Chem. 1993,40, 1-88 (10) (a) Johnson, J. L.; Hainline, B. E.; Rajagopalan, K. V. J. Biol. Chem. f 980,255, 1783. (b) Johnson, J. L.; Hainline, B. E.; Rajagopalan, K. V.; Arison, B. H. J . Biol. Chem. 1984, 259, 5414. (11) (a) Johnson, 3. L.; Rajagopalan, K. V. Proc. Natl. Acad. Sci. USA. 1982, 79, 6856. (b) Rajagopalan, K. V. Adv. Enzymol. Relat. Areas Mol. Biol. 1991, 64, 215-289. (12) Chan, M. K.; Mukund, S.; Lketzin, A.; Adams, M. W. W.; Rees, D. C. Science 1995, 267, 1463-1469. (13) (a) Davis, M. D.; Edmondson, D. E.; Muller, F. Eur. J . Biochem. 1984,145,237. (b) Edmondson, D. E.; Davis, M. D.; Muller, F. In Flavins and Flavoproteins; Bray, R. C . , Engel, P. C., Mayhew, S. G., Eds.; Walter de Gruyter: Berlin, 1984; p 309. (14) Kiisthardt, U.; LaBarre, M. J.; Enemark, J. H. Znorg. Chem. 1990, 29, 3182. (15) Mims, W. B. Phys. Rev. 19’72, B5, 2409; 1972, B6, 3543. (16) Romanelli, M.; Kevan, L. Appl. Magn. Reson. 1992,3, 1079, 1099. (17) Cleland, W. E., Jr.; Barnhart, K. M.; Yamanouchi, K.; Collison, D.; Mabbs, F. E.; Ortega, R. B.; Enemark, J. H. Znorg. Chem. 1987, 26, 1017-1025. (18) Chan, M. K.; Rees, D. C. Private communication.
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