Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 1794-1797, May 1977 Chemistry
X-ray absorption studies of halide binding to carbonic anhydrase (metalloenzymes/EXAFS/protein structure)
G. S. BROWN, G. NAVON*, AND R. G. SHULMAN Bell Laboratories, Murray Hill, New Jersey 07974
Contributed by R. G. Shulman, March 9, 1977
ABSTRACT X-ray absorption measurements of bovine carbonic anhydrase B have been made at the Stanford Synchrotron Radiation Project with a spectrometer operating in the fluorescence mode. Differences in absorption at and beyond the zinc K-edge near 9664 eV have been observed upon the addition of bromide or iodide. The additional absorption in k space, out to k 7 A-i, obtained upon the addition of iodide has been compared with the absorption in this region of a ZnI2 sample. The similarities between these absorptions lead to the conclusion that the zinc-iodide distance in the protein is 2.65 ± 0.06 A; it is known to be 2.62 A in ZnI2. This shows that the iodide binds directly to the zinc in the protein.
metal-ligand distances accurately in a protein, we have made these measurements on iodide complexes of zinc bovine carbonic anhydrase B and have been able to show that the iodide is, in fact, bound directly to the zinc. MATERIALS Bovine carbonic anhydrase B, chromatographically prepared by the method of Lindskog (26), was purchased from Miles Laboratories. To an unbuffered 5 mM solution of the enzyme at pH 6.0, NaBr and NaI were added to final concentrations of 0.5 and 0.1 M, respectively. These concentrations are about 10-fold higher than the reported dissociation constants of the enzyme-anion complexes (27). The solutions were then lyophilized. Anhydrous ZnI2 was purchased from Fisher Chemical Co.
Synchrotron radiation has recently provided high-intensity sources for x-ray absorption measurements (1-4) which have allowed interpretable spectra to be obtained from dilute absorbers, in particular from the metal atoms in metalloproteins. In the iron proteins rubredoxin (5, 6) and hemoglobin (7, 8), distances to the known ligands of the iron have been determined with great accuracy. Another question that might be answerable by x-ray absorption measurements asks, What are the ligands of a metal ion in a particular metalloprotein? This question is usually answered definitively by an x-ray diffraction determination of the protein structure combined with knowledge of the amino acid sequence. An x-ray crystallographic structure determination has in fact been made of the zinc protein human carbonic anhydrase C (carbonate hydro-lyase, EC 4.2.1.1), with a resolution of 2.0 A (9). This enzyme catalyzes the reaction (10) H+ + HCO3- T H20 + CO2. Hence, in addition to the protein structure it is also important to understand the binding of anions because they serve as models for the HCO3- substrate and are also known to be competitive inhibitors of the enzyme in vitro (10-14) as well as in vivo. Changes in the optical spectra (15, 16) when anions bound to the Co(II) derivative of carbonic anhydrase indicated they were ligands of the metal. For this reason it was disturbing that the crystal structure of human carbonic anhydrase C (17), determined in this case with a resolution of 2.5 A, showed that the iodide ion was 3.5-3.7 A from the zinc. This unusual distance raised the question as to whether the iodide was bound directly to the zinc or perhaps inhibited anion binding by steric hindrance from a nearby iodide binding site. In contrast to this large separation, there was evidence for direct halide binding to the zinc from the broadening of the s5CI nuclear magnetic resonance lines observed by Ward (18-20). Additional support for the direct anion binding has been obtained from electron paramagnetic resonance studies of the Co(II) enzyme (21, 22) and various nuclear magnetic resonance studies (23-25) on bovine carbonic anhydrase B. Because x-ray absorption spectroscopy can determine
METHODS The atomic x-ray absorption coefficient, ,u(E), exhibits sharp peaks within 10-20 eV of a K or L absorption edge and an oscillatory modulation up to 1000 eV above the absorption edge. The structure in the vicinity of the edge corresponds to transitions to bound states and has been shown to be extremely sensitive to the ligands, valence state, and point symmetry of the target atom (28). The oscillatory modulation is caused by the back-scattering of the photoelectron, which introduces an energy-dependent modification of the outgoing wavefunction of the photoelectron. A straightforward treatment of this process, regarding the neighboring atoms as point scatterers with complex electron backscattering amplitude f(k, r), yields the following expression for the oscillatory modulation of the absorption cross section (2, 29, 30):
'(k) = Z
Nif1(k,r) sin[2kRj
a(k)] exp (- 2) exp(-2aj2k2) [1] in which k is the photoelectron wave vector, Nj is the number of atoms in the jth shell at distance Rp, a(k) is the sum of central atom and ligand phase shifts, u2 is the mean square relative displacement from equilibrium, and X is the photoelectron mean free path. Experimental (31) studies have shown that a(k) is transferable among identical atomic pairs while more recently theoretical values of a(k) have been used to determine values of Rj with comparable accuracy (32). At the present time, the most thoroughly tested practice is to measure the phase function a(k) for model compounds with ligands identical to the ligands +
of the unknown and to use this value to determine the unknown distances. The experiments described in this paper were performed on the EXAFS I x-ray beam line at the Stanford Synchrotron Radiation Project. The x-ray monochromator and beam transport system have been described in detail elsewhere (4). The x-ray absorption was measured by the fluorescence technique (33)
* Present address: Department of Chemistry, Tel Aviv University, Ramat-Aviv, Tel-Aviv, Israel.
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Proc. Natl. Acad. Sci. USA 74 (1977)
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(eV) FIG. 1. X-ray absorption of different complexes of bovine carbonic anhydrase as a function of energy in the vicinity of the zinc-Ka edge. The solid curve was observed in the control sample without halide ions; the dashed curve was for the bromide complex; and the dotted curve was for the iodide complex.
which is about an order of magnitude more sensitive then the transmission method for typical metalloenzymes. The fluorescence spectrometer was a nine-element array of NaI detectors, each designed to count at a rate of 200,000 Hz without significant saturation. The spectrometer, oriented at 90° to the incident beam and in the horizontal plane, registered KL and KM fluorescence x-rays from the zinc ions (8638 and 9571 eV, respectively) as well as Compton-Rayleigh scattered protons. The scattered radiation is weakest at 900 from the incident beam in the horizontal plane, because of the strong linear polarization of the x-ray beam. However, because of the large polar angle spanned by the spectrometer (about +400), each detector has a unique background rate, determined by its angular position, and a unique signal-to-noise ratio. Therefore, a weighted sum was performed on-line to maximize the overall signal-to-noise ratio. Data were accumulated at energy intervals of 0.3 eV for studies of the absorption edge structure and at intervals of 1.0 eV for the EXAFS region. The dwell time was 1.0 sec per point in all cases, and each scintillation counter operated at a rate between 100 and 200 kHz. It should also be remembered that the data were accumulated in intervals uniformly spaced in monochromator angle; to be interpreted in terms of Eq. 1, the data were first converted to units of energy and then to units of the electron wave vector k. Furthermore, because the model compound data were accumulated on a separate run after the enzyme measurements had been completed, the spectrometer had to be recalibrated by aligning the characteristic sharp "glitches" in the monochromator output. This technique permits a relative calibration to a precision of ±1 data point, which is well within the uncertainty in the choice of the edge position.
RESULTS Fig. 1 compares the absorption edges, over a range of 100 V, of three different complexes of carbonic anhydrase. The control sample presumably had H20 as the fourth zinc ligand. The vertical heights have been adjusted so that the first strong peaks have the same height. On the basis of previous studies of the edge absorption in transition metal complexes, these peaks arise from transitions of the Is electron to bound states (28). It is clear from these spectra that there are sizable differences in these samples, which most likely arise from the ligands of the zinc. However, without specific assignments of the peaks, we cannot be sure that the differences do not arise from different contri-
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FIG. 2. The modulation of the x-ray absorption has been multiplied by k and plotted in k space after the background was removed as described in the text. The solid curve is for the iodide complex of carbonic anhydrase; the dashed line is for the control, in which water is presumably the fourth zinc ligand.
butions to the EXAFS spectra, near the edge, from different nonbonded neighbors. These reservations are removed by analysis of the EXAFS spectra. In Fig. 2 we compare the EXAFS spectra of carbonic anhydrase with and without iodide. The data have been transformed to k space, multiplied by k, and smoothed to eliminate the high-frequency noise introduced by the initial choice of the number of data channels. The background has been removed by a cubic spline fit by using a technique developed by B. Kincaid (personal communication). Although in many respects the spectra are similar, there are significant differences in amplitudes and positions considerably above the noise for k < 7 A-1 consistent with some of the zinc ligands being the same and some different in the two compounds. A difference spectrum between the iodide complex and the control was obtained by subtracting the two raw data sets, in order to minimize the possibility of contributions from the background removal procedure. In Fig. 3 this difference spectrum is superimposed upon the ZnI2 spectrum, filtered to remove all but the zinciodide bond contributions. It is important to notice that, by taking the difference between the two proteins' absorptions, we decreased the contribution of the nitrogen (and oxygen) ligands relative to that of the iodide. Hence, in R space it was possible to select the zinciodide contribution by filtering, because it was not on the shoulder of a larger peak from the nitrogens. It is apparent from Fig. 3 that the zinc-iodide distance is approximately the same in the enzyme and in ZnI2. We have refined this estimate by applying a similar Fourier filter to the protein difference spectrum and the spectrum of ZnI2. The filtered protein difference spectrum was multiplied by a slowly varying amplitude function to equalize the amplitudes at all k values. The phase function of the resulting sinusoid was then computed, and a linear function, 2 kAR, with variable AR, was added to the ZnI2 phase function to bring the two functions into coincidence, according to the least squares criterion. Simultaneously, the photoelectron energy threshold of the unknown, Ea was varied to force the two functions to have the same phase at k = 0. This is justified by the observation that the photoelectron absorption edge position can shift by several volts with the valence state of the central atom. This procedure has been developed by B. Kincaid (personal communication). In this way we calculated that. Renzyme- Rmodel = 0.03 ± 0.06 A. From crystallographic (34) measurements of ZnI2, Rmodei = 2.62 A; consequently, Renzyme = 2.65 ± 0.06 A. The uncertainty quoted is double the uncertainty ascribed to the least squares fit and
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k (A-) FIG. 3. The dashed curve is the absorption of ZnI2 which has been Fourier-transformed to R space, filtered to retain the ZnI2 contributions, and retransformed back into k space, as described in the text. The solid curve is the filtered difference between the two spectra of Fig. 2 and, as such, represents the zinc-iodide contributions to the absorption plotted in k space.
is our best estimate of the limits of systematic errors. It should be remarked that an error of 0.06 A corresponds to a phase shift of ir/4 at k = 6.8, which is certainly larger than is allowed by the data shown in Fig. 3.
DISCUSSION Zinc(II) ions are present in the active sites of many metalloenThey are sometimes referred to as "silent" metal ions because they do not exhibit optical absorption spectra in the visible and the near-UV and, being diamagnetic, do not give an electron paramagnetic resonance signal. Therefore, in order to probe their environment, it is often necessary to replace them with other transition metal ions such as Co(II) or Mn(II). The present study demonstrates that by the use of x-ray absorption it is possible to identify and locate an iodide ion as a ligand to the zinc(II) ion in the active site of bovine carbonic anhydrase B. This technique should be applicable to other zinc metalloenzymes. Our finding that the iodide ion is bound in the first coordination shell of the zinc(II) ion in the active site of bovine carbonic anhydrase B agrees with the -`Cl nuclear magnetic resonance result, in which line broadening caused by carbonic anhydrase was taken as an evidence for direct binding of the chloride ion to the zinc(II) (18-20). In similar studies using 81Br, line broadening could be observed in the presence of bovine carbonic anhydrase (35) but not for human carbonic anhydrase B (35, 36), presumably due to slow exchange of the bromide ion. Our results are in contrast to the x-ray diffraction data on a single crystal of human carbonic anhydrase C in which zinciodide distances of 3.5-3.7A were reported (17). Bovine carbonic anhydrase B and human carbonic anhydrase C are both zymes.
Proc. Natl. Acad. Sci. USA 74 (1977)
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high-activity forms of the enzyme and have many similar characteristics (37, 38). Therefore a similar mode of iodide binding is expected. Many of the properties of carbonic anhydrase depend on the group in the active site which titrates around neutral pH and whose basic form is catalytically active. It is the acidic form that is responsible for the binding of inhibitory anions. The nature of the acid-base transformation at the active site is not yet fully understood, partly because of the inability to obtain single crystals of carbonic anhydrase at acidic pH. Consequently, the x-ray diffraction data showing the iodide binding were obtained from the basic form of the enzyme, which normally does not bind anions. Although we cannot rule out possible differences between human carbonic anhydrase C and bovine carbonic anhydrase B, we can say, from the present results, that a normal zinc-iodide bond exists in the latter. Binding of two acetate and two azide anions has been observed for zinc and manganese bovine carbonic anhydrase B (23, 24). For the manganese enzyme the distance of the methyl protons of the more weakly bound, inhibitory acetate ion from the metal ion was found to be 4.3 A, consistent with direct binding of the carboxylate group to the metal ion. On the other hand, the more tightly bound noninhibitory acetate ion was farther from the metal ion and was postulated to bind at the iodide site found in the x-ray. Similar results were more recently found by 13C nuclear magnetic resonance studies for the binding of bicarbonate anion to Co(II) bovine carbonic anhydrase (25). Again an inhibitory, weakly bound bicarbonate was found to be directly bound to the metal ion and a noninhibitory one was more tightly bound but farther away from the metal ion. Because the bicarbonate ion is the substrate of the enzyme, the two bound ions are implicated in the catalytic mechanism of the enzyme (25). The question of whether two iodide ions are bound simultaneously in the active site of carbonic anhydrase remains open because the photoelectron scattering by the second iodide is expected to be considerably weaker and probably would not be identifiable in the present experiments. It is possible that the second binding site for the iodide ion found by x-ray diffraction of single crystals of the basic form of the enzyme is the second binding site of the acetate, azide, and bicarbonate anions. However, it is clear from our results that there is one iodide site 2.65 i 0.06 A from the zinc, which is a normal bond distance. We thank Drs. P. Eisenberger and B. M. Kincaid for many helpful conversations. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
1. de L. Kronig, R. (1931) Physik 70,317-323. 2. Sayers, D. E., Lytle, F. W. & Stern, E. A. (1970) Advances in
3. 4.
5. 6. 7. 8.
X-ray Analysis (Plenum Press, New York), Vol. 13, pp. 248271. Kincaid, B. M. & Eisenberger, P. (1975) Phys. Rev. Lett. 34, 1361-1364. Kincaid, B. M., Eisenberger, P. & Sayers, D. E. (1977) Phys. Rev., in press. Shulman, R. G., Eisenberger, P., Blumberg, W. E. & Stombaugh, N. A. (1975) Proc. Natl. Acad. Sci. USA 72,4003-4007. Sayers, D. E., Stern, E. A. & Herriott, X. (1976) J. Chem. Phys. 64, 427-428. Kincaid, B. M., Eisenberger, P., Hodgson, K. 0. & Doniach, S. (1975) Proc. Natl. Acad. Sci. USA 72, 2340-2342. Eisenberger, P. M., Shulman, R. G., Brown, G. S. & Ogawa, S.
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(1976) Proc. Natl. Acad. Sci. USA 73,491-495. 9. Kannan, K. K., Liljas, A., Waara, I., Bergsten, P.-C., Lovgren, S., Strandberg, B., Bengtsson, U., Carlbom, U., Fridborg, K., Jarup, L. & Petef, M. (1971) Cold Spring Harbor Symp. Quant. Biol. 36,221-231. 10. Roughton, F. J. W. & Booth, V. H. (1946) Biochem. J. 40, 309-330. 11. Kernohan, J. C. (1965) Biochim. Biophys. Acta 96,304-317. 12. Maren, T. H., Rayburn, C. S. & Liddle, N. E. (1976) Science 191, 469-472. 13. Koenig, S. H. & Brown, R. D., III (1976) Science 194, 745746. 14. Maren, T. H. (1976) Science 194, 746-747. 15. Lindskog, S. (1963) J. Biol. Chem. 238,945-951. 16. Linkskog, S. (1966) Biochemistry 5, 2641-2646. 17. Bergsten, P.-C., Waara, I., Lovgren, S., Liljas, A., Kannan, K. K. & Bengtsson, U. (1972) in Oxygen Affinity of Hemoglobin and Rec Cell Acid-Base Status, Alfred Benzon Symposium IV (Munksgaard, Copenhagen and Academic Press, New York), pp. 363-383. 18. Ward, R. L. (1969) Biochemistry 8, 1879-1883. 19. Ward, R. L. (1970) Biochemistry 9,2447-2454. 20. Ward, R. L. & Cull, M. D. (1972) Arch. Biochem. Biophys. 150, 436-439. 21. Taylor, J. S. & Coleman, J. E. (1971) J. Biol. Chem. 246, 7058-7067. 22. Grell, E. & Bray, R. C. (1971) Biochim. Biophys. Acta 236, 503-506.
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23. Lanir, A. & Navon, G. (1974) Biochim. Biophys. Acta 341, 65-74. 24. Lanir, A. & Navon, G. (1974) Biochim. Biophys. Acta 341, 75-84. 25. Yeagle, P. L., Lochmuller, C. H. & Henkens, R. W. (1975) Proc. Natl. Acad. Sci. USA 72, 454-458. 26. Lindskog, S. (1960) Biochim. Biophys. Acta 39,218-226. 27. Pocker, Y. & Stone, J. T. (1968) Biochemistry 7, 2936-2945. 28. Shulman, R. G., Yafet, Y., Eisenberger, P. & Blumberg, W. E. (1976) Proc. Natl. Acad. Sci. USA 73, 1384-1388. 29. Ashley, C. A. & Doniach, S. (1975) Phys. Rev. [Sect. B] 11, 1279-1288. 30. Lee, P. A. & Pendry, J. B. (1975) Phys. Rev. [Sect. B] 11, 2795-2811. 31. Citrin, P. H., Eisenberger, P. & Kincaid, B. M. (1976) Phys. Rev. Lett. 36, 1346-1349. 32. Lee, P. A. & Beni, G. (1977) Phys. Rev. [Sect. B], in press. 33. Jaklevic, J., Kirby, J. A., Klein, M. P., Robertson, A. S., Brown, G. S. & Eisenberger, P. (1977) Solid State Commun., in press. 34. Oswald, H. R. (1960) Helv. Chim. Acta 43,77-80. 35. Ward, R. L. & Whitney, P. L. (1973) Biochim. Biophys. Res. Commun. 151,343-348. 36. Zeppezauer, M., Lindman, B., Forsen, S. & Lindqvist, I. (1969) Biochem. Biophys. Res. Commun. 37, 137-142. 37. Maren, T. H. (1967) Phys. Rev. 47,595. 38. Lindskog, S., Henderson, L. E., Kannan, K. K., Lilias, A., Nyman, P. 0. & Strandberg, B. (1971) in The Enzymes, ed. Boyer, P. D. (Academic Press, New York), 3rd ed., Vol. 5, p. 587.
X-ray absorption studies of halide binding to carbonic anhydrase (metalloenzymes/EXAFS/protein structure)
G. S. BROWN, G. NAVON*, AND R. G. SHULMAN Bell Laboratories, Murray Hill, New Jersey 07974
Contributed by R. G. Shulman, March 9, 1977
ABSTRACT X-ray absorption measurements of bovine carbonic anhydrase B have been made at the Stanford Synchrotron Radiation Project with a spectrometer operating in the fluorescence mode. Differences in absorption at and beyond the zinc K-edge near 9664 eV have been observed upon the addition of bromide or iodide. The additional absorption in k space, out to k 7 A-i, obtained upon the addition of iodide has been compared with the absorption in this region of a ZnI2 sample. The similarities between these absorptions lead to the conclusion that the zinc-iodide distance in the protein is 2.65 ± 0.06 A; it is known to be 2.62 A in ZnI2. This shows that the iodide binds directly to the zinc in the protein.
metal-ligand distances accurately in a protein, we have made these measurements on iodide complexes of zinc bovine carbonic anhydrase B and have been able to show that the iodide is, in fact, bound directly to the zinc. MATERIALS Bovine carbonic anhydrase B, chromatographically prepared by the method of Lindskog (26), was purchased from Miles Laboratories. To an unbuffered 5 mM solution of the enzyme at pH 6.0, NaBr and NaI were added to final concentrations of 0.5 and 0.1 M, respectively. These concentrations are about 10-fold higher than the reported dissociation constants of the enzyme-anion complexes (27). The solutions were then lyophilized. Anhydrous ZnI2 was purchased from Fisher Chemical Co.
Synchrotron radiation has recently provided high-intensity sources for x-ray absorption measurements (1-4) which have allowed interpretable spectra to be obtained from dilute absorbers, in particular from the metal atoms in metalloproteins. In the iron proteins rubredoxin (5, 6) and hemoglobin (7, 8), distances to the known ligands of the iron have been determined with great accuracy. Another question that might be answerable by x-ray absorption measurements asks, What are the ligands of a metal ion in a particular metalloprotein? This question is usually answered definitively by an x-ray diffraction determination of the protein structure combined with knowledge of the amino acid sequence. An x-ray crystallographic structure determination has in fact been made of the zinc protein human carbonic anhydrase C (carbonate hydro-lyase, EC 4.2.1.1), with a resolution of 2.0 A (9). This enzyme catalyzes the reaction (10) H+ + HCO3- T H20 + CO2. Hence, in addition to the protein structure it is also important to understand the binding of anions because they serve as models for the HCO3- substrate and are also known to be competitive inhibitors of the enzyme in vitro (10-14) as well as in vivo. Changes in the optical spectra (15, 16) when anions bound to the Co(II) derivative of carbonic anhydrase indicated they were ligands of the metal. For this reason it was disturbing that the crystal structure of human carbonic anhydrase C (17), determined in this case with a resolution of 2.5 A, showed that the iodide ion was 3.5-3.7 A from the zinc. This unusual distance raised the question as to whether the iodide was bound directly to the zinc or perhaps inhibited anion binding by steric hindrance from a nearby iodide binding site. In contrast to this large separation, there was evidence for direct halide binding to the zinc from the broadening of the s5CI nuclear magnetic resonance lines observed by Ward (18-20). Additional support for the direct anion binding has been obtained from electron paramagnetic resonance studies of the Co(II) enzyme (21, 22) and various nuclear magnetic resonance studies (23-25) on bovine carbonic anhydrase B. Because x-ray absorption spectroscopy can determine
METHODS The atomic x-ray absorption coefficient, ,u(E), exhibits sharp peaks within 10-20 eV of a K or L absorption edge and an oscillatory modulation up to 1000 eV above the absorption edge. The structure in the vicinity of the edge corresponds to transitions to bound states and has been shown to be extremely sensitive to the ligands, valence state, and point symmetry of the target atom (28). The oscillatory modulation is caused by the back-scattering of the photoelectron, which introduces an energy-dependent modification of the outgoing wavefunction of the photoelectron. A straightforward treatment of this process, regarding the neighboring atoms as point scatterers with complex electron backscattering amplitude f(k, r), yields the following expression for the oscillatory modulation of the absorption cross section (2, 29, 30):
'(k) = Z
Nif1(k,r) sin[2kRj
a(k)] exp (- 2) exp(-2aj2k2) [1] in which k is the photoelectron wave vector, Nj is the number of atoms in the jth shell at distance Rp, a(k) is the sum of central atom and ligand phase shifts, u2 is the mean square relative displacement from equilibrium, and X is the photoelectron mean free path. Experimental (31) studies have shown that a(k) is transferable among identical atomic pairs while more recently theoretical values of a(k) have been used to determine values of Rj with comparable accuracy (32). At the present time, the most thoroughly tested practice is to measure the phase function a(k) for model compounds with ligands identical to the ligands +
of the unknown and to use this value to determine the unknown distances. The experiments described in this paper were performed on the EXAFS I x-ray beam line at the Stanford Synchrotron Radiation Project. The x-ray monochromator and beam transport system have been described in detail elsewhere (4). The x-ray absorption was measured by the fluorescence technique (33)
* Present address: Department of Chemistry, Tel Aviv University, Ramat-Aviv, Tel-Aviv, Israel.
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(eV) FIG. 1. X-ray absorption of different complexes of bovine carbonic anhydrase as a function of energy in the vicinity of the zinc-Ka edge. The solid curve was observed in the control sample without halide ions; the dashed curve was for the bromide complex; and the dotted curve was for the iodide complex.
which is about an order of magnitude more sensitive then the transmission method for typical metalloenzymes. The fluorescence spectrometer was a nine-element array of NaI detectors, each designed to count at a rate of 200,000 Hz without significant saturation. The spectrometer, oriented at 90° to the incident beam and in the horizontal plane, registered KL and KM fluorescence x-rays from the zinc ions (8638 and 9571 eV, respectively) as well as Compton-Rayleigh scattered protons. The scattered radiation is weakest at 900 from the incident beam in the horizontal plane, because of the strong linear polarization of the x-ray beam. However, because of the large polar angle spanned by the spectrometer (about +400), each detector has a unique background rate, determined by its angular position, and a unique signal-to-noise ratio. Therefore, a weighted sum was performed on-line to maximize the overall signal-to-noise ratio. Data were accumulated at energy intervals of 0.3 eV for studies of the absorption edge structure and at intervals of 1.0 eV for the EXAFS region. The dwell time was 1.0 sec per point in all cases, and each scintillation counter operated at a rate between 100 and 200 kHz. It should also be remembered that the data were accumulated in intervals uniformly spaced in monochromator angle; to be interpreted in terms of Eq. 1, the data were first converted to units of energy and then to units of the electron wave vector k. Furthermore, because the model compound data were accumulated on a separate run after the enzyme measurements had been completed, the spectrometer had to be recalibrated by aligning the characteristic sharp "glitches" in the monochromator output. This technique permits a relative calibration to a precision of ±1 data point, which is well within the uncertainty in the choice of the edge position.
RESULTS Fig. 1 compares the absorption edges, over a range of 100 V, of three different complexes of carbonic anhydrase. The control sample presumably had H20 as the fourth zinc ligand. The vertical heights have been adjusted so that the first strong peaks have the same height. On the basis of previous studies of the edge absorption in transition metal complexes, these peaks arise from transitions of the Is electron to bound states (28). It is clear from these spectra that there are sizable differences in these samples, which most likely arise from the ligands of the zinc. However, without specific assignments of the peaks, we cannot be sure that the differences do not arise from different contri-
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FIG. 2. The modulation of the x-ray absorption has been multiplied by k and plotted in k space after the background was removed as described in the text. The solid curve is for the iodide complex of carbonic anhydrase; the dashed line is for the control, in which water is presumably the fourth zinc ligand.
butions to the EXAFS spectra, near the edge, from different nonbonded neighbors. These reservations are removed by analysis of the EXAFS spectra. In Fig. 2 we compare the EXAFS spectra of carbonic anhydrase with and without iodide. The data have been transformed to k space, multiplied by k, and smoothed to eliminate the high-frequency noise introduced by the initial choice of the number of data channels. The background has been removed by a cubic spline fit by using a technique developed by B. Kincaid (personal communication). Although in many respects the spectra are similar, there are significant differences in amplitudes and positions considerably above the noise for k < 7 A-1 consistent with some of the zinc ligands being the same and some different in the two compounds. A difference spectrum between the iodide complex and the control was obtained by subtracting the two raw data sets, in order to minimize the possibility of contributions from the background removal procedure. In Fig. 3 this difference spectrum is superimposed upon the ZnI2 spectrum, filtered to remove all but the zinciodide bond contributions. It is important to notice that, by taking the difference between the two proteins' absorptions, we decreased the contribution of the nitrogen (and oxygen) ligands relative to that of the iodide. Hence, in R space it was possible to select the zinciodide contribution by filtering, because it was not on the shoulder of a larger peak from the nitrogens. It is apparent from Fig. 3 that the zinc-iodide distance is approximately the same in the enzyme and in ZnI2. We have refined this estimate by applying a similar Fourier filter to the protein difference spectrum and the spectrum of ZnI2. The filtered protein difference spectrum was multiplied by a slowly varying amplitude function to equalize the amplitudes at all k values. The phase function of the resulting sinusoid was then computed, and a linear function, 2 kAR, with variable AR, was added to the ZnI2 phase function to bring the two functions into coincidence, according to the least squares criterion. Simultaneously, the photoelectron energy threshold of the unknown, Ea was varied to force the two functions to have the same phase at k = 0. This is justified by the observation that the photoelectron absorption edge position can shift by several volts with the valence state of the central atom. This procedure has been developed by B. Kincaid (personal communication). In this way we calculated that. Renzyme- Rmodel = 0.03 ± 0.06 A. From crystallographic (34) measurements of ZnI2, Rmodei = 2.62 A; consequently, Renzyme = 2.65 ± 0.06 A. The uncertainty quoted is double the uncertainty ascribed to the least squares fit and
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k (A-) FIG. 3. The dashed curve is the absorption of ZnI2 which has been Fourier-transformed to R space, filtered to retain the ZnI2 contributions, and retransformed back into k space, as described in the text. The solid curve is the filtered difference between the two spectra of Fig. 2 and, as such, represents the zinc-iodide contributions to the absorption plotted in k space.
is our best estimate of the limits of systematic errors. It should be remarked that an error of 0.06 A corresponds to a phase shift of ir/4 at k = 6.8, which is certainly larger than is allowed by the data shown in Fig. 3.
DISCUSSION Zinc(II) ions are present in the active sites of many metalloenThey are sometimes referred to as "silent" metal ions because they do not exhibit optical absorption spectra in the visible and the near-UV and, being diamagnetic, do not give an electron paramagnetic resonance signal. Therefore, in order to probe their environment, it is often necessary to replace them with other transition metal ions such as Co(II) or Mn(II). The present study demonstrates that by the use of x-ray absorption it is possible to identify and locate an iodide ion as a ligand to the zinc(II) ion in the active site of bovine carbonic anhydrase B. This technique should be applicable to other zinc metalloenzymes. Our finding that the iodide ion is bound in the first coordination shell of the zinc(II) ion in the active site of bovine carbonic anhydrase B agrees with the -`Cl nuclear magnetic resonance result, in which line broadening caused by carbonic anhydrase was taken as an evidence for direct binding of the chloride ion to the zinc(II) (18-20). In similar studies using 81Br, line broadening could be observed in the presence of bovine carbonic anhydrase (35) but not for human carbonic anhydrase B (35, 36), presumably due to slow exchange of the bromide ion. Our results are in contrast to the x-ray diffraction data on a single crystal of human carbonic anhydrase C in which zinciodide distances of 3.5-3.7A were reported (17). Bovine carbonic anhydrase B and human carbonic anhydrase C are both zymes.
Proc. Natl. Acad. Sci. USA 74 (1977)
Brown et al.
high-activity forms of the enzyme and have many similar characteristics (37, 38). Therefore a similar mode of iodide binding is expected. Many of the properties of carbonic anhydrase depend on the group in the active site which titrates around neutral pH and whose basic form is catalytically active. It is the acidic form that is responsible for the binding of inhibitory anions. The nature of the acid-base transformation at the active site is not yet fully understood, partly because of the inability to obtain single crystals of carbonic anhydrase at acidic pH. Consequently, the x-ray diffraction data showing the iodide binding were obtained from the basic form of the enzyme, which normally does not bind anions. Although we cannot rule out possible differences between human carbonic anhydrase C and bovine carbonic anhydrase B, we can say, from the present results, that a normal zinc-iodide bond exists in the latter. Binding of two acetate and two azide anions has been observed for zinc and manganese bovine carbonic anhydrase B (23, 24). For the manganese enzyme the distance of the methyl protons of the more weakly bound, inhibitory acetate ion from the metal ion was found to be 4.3 A, consistent with direct binding of the carboxylate group to the metal ion. On the other hand, the more tightly bound noninhibitory acetate ion was farther from the metal ion and was postulated to bind at the iodide site found in the x-ray. Similar results were more recently found by 13C nuclear magnetic resonance studies for the binding of bicarbonate anion to Co(II) bovine carbonic anhydrase (25). Again an inhibitory, weakly bound bicarbonate was found to be directly bound to the metal ion and a noninhibitory one was more tightly bound but farther away from the metal ion. Because the bicarbonate ion is the substrate of the enzyme, the two bound ions are implicated in the catalytic mechanism of the enzyme (25). The question of whether two iodide ions are bound simultaneously in the active site of carbonic anhydrase remains open because the photoelectron scattering by the second iodide is expected to be considerably weaker and probably would not be identifiable in the present experiments. It is possible that the second binding site for the iodide ion found by x-ray diffraction of single crystals of the basic form of the enzyme is the second binding site of the acetate, azide, and bicarbonate anions. However, it is clear from our results that there is one iodide site 2.65 i 0.06 A from the zinc, which is a normal bond distance. We thank Drs. P. Eisenberger and B. M. Kincaid for many helpful conversations. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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