EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN

Transcription

1 EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN S. Hasnain, L. Alagna, N. Blackburn, R. Strange To cite this version: S. Hasnain, L. Alagna, N. Blackburn, R. Strange. EXAFS AND XANES OF SUPEROXIDE DISMU- TASE : A COPPER PROTEIN. Journal de Physique Colloques, 1986, 47 (C8), pp.c c < /jphyscol: >. <jpa > HAL Id: jpa Submitted on 1 Jan 1986 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 JOURNAL DE PHYSIQUE Colloque C8, suppl6ment au no 12, Tome 47, d6cembre 1986 EXAFS AND XANES OF SUPEROXIDE DISMUTASE : A COPPER PROTEIN S.S. HAsNAIN*, L. ALAGNA**, N.J. BLACKBURN*** and R.W. STRANGE*,"' "synchrotron Radiation Source, SERC Daresbury Laboratory, GB-Warrington WA4 4AD, Great-Britain Consiglio Nazionale delle Ricerche, Via Salaria, KM , Casella Postale 10, Monterotondo Stazione, Roma, Italy ""~epartment of Chemistry, UMIST, P.O. Box 88, GB-Manchester M60 lqv, Great-Britain Abstract EXAFS and XANES have provided uriique and specific structural information for a number of copper proteins. Some of this work has recently been reviewed ill. We report here the EXAFS and XANES work on superoxide dismutase in some detail to highlight some of the structural information which EXAFS and XANES have provided, and also to report on some of the recent developments for calculating multiple scattering effects in the EXAFS region. An attempt has been made to understand the high resolution XANES region of superoxide dismutase, which shows the presence of interligand scattering in addition to the strong multiple scattering from the atoms of an imidazole ring [21. This offers an exciting possibility of investigating the stereochemical changes which accompany ligand binding or reduction of the protein. 1. EXAFS and XANES The x-ray absorption spectrum has been traditionally divided into an EXAFS and a XANES region; structure observed at some ev above the absorption threshold has been referred to as the EXAFS region while structure below it as the XANES. As we shall see in the following section, the EXAFS region can be extended to lower values of photoelectron energies if use is made of the spherical wave method and the multiple scattering effects are considered from atoms of a ring such as an imidazole. At high photoelectron energies, the simplified plane wave approximation for EXAFS is valid [3 I. At lower electron energies, the curvature of the electron wave cannot be neglected and thus the plane wave approximation breaks down and leads to incorrect determination of interatomic distances. Furthermore, it is this low energy part which contains most of the information, particularly from the more distant shell. Also, for metalloproteins where the scatterers are generally weak (0, N etc), the data is most reliable at lower energies. The low energy part of the EXAFS spectrum can be used if use of the exact theory given by Lee and Pendry [41 is made, which takes account of the curvature of electron wave and thus has been namcd the 'curved wave method'. Article published online by EDP Sciences and available at

3 JOURNAL DE PHYSIQUE In general this single scattering theory is sufficient for interpretation of EXAFS data. However, strong multiple scattering effects can result from geometrical arrangement of ligands e.g. in an imidazole ligand strong multiple scattering exists due to an almost collinear geometry of Cu-Na-C. This has been referred to as a 'shadowing1 or sometimes 'focusing' effect andyhas been suggested to cause a shortening of the Cu-C distance when the low energy part of the EXAFS spectrum is fitted [5]. This shortexin9 of the bond results from a phase modification due to the intervening atom. Recently the single scattering curved-wave EXAFS function has been modified 161 to include the double- and triple-scattering contributions, which are expected to be the only other contributions of significance in the EXAFS region. These multiple scattering contributions and higher order scattering become even more important in the x-ray absorption near-edge region (XANES) [71. In the XANES, multiple scattering contributions are strong even in the open structure and thus offer a possibility of obtaining a fuller stereochemical picture of the metal site in proteins. Recent theoretical developments have made this region of the spectrum tractable to theoretical interpretation and offers a unique method of obtaining higher correlation functions in aqueous protein samples. 2. Superoxide dismutase ( Cu-Zn) A great deal of structural information is available for the bovine Cu-Zn SOD; including its amino acid sequence, and the crystal structure determination at high resolution [a]. It is a dimeric protein, of molecular weight - 32,000 dalton, with each of the subunits containing a Cu and Zn atom. The crystal structure of the bovine superoxide dismutase suggests that the subunits are held together by non-covalent interactions. Within the subunit, the Cu(1I) and!&(i1 ) ions are observed to be separated by and bridged by the imidazole ring of a histidine (His-61) residue, which is believed to be deprotonated. The Zn ion is coordinated to the nitrogen atoms of each of the imidazole rings of histidine residues 61, 69 and 78 and an oxygen atom (carboxylate) of the aspartate 82. All of the Zn ligands are derived from one continuous piece of the polypeptide chain, which is not the case for the Cu(I1) ion. The crystal structure shows that Cu(I1) ion is coordinated to four imidazole rings from histidines 44, 46, 61 and Protein solution and freeze-dried powder Blackburn et al have used the EXAFS and XANES technique for studying the Cu and Zn sites of the freeze-dried powder 151 and solution [I 03 Cu-Zn bovine superoxide dismutase. They were also able to show that the 4 Cu-N distance for the protein was A, which is typical for [C~(imid)~]*+ complexes. At the time the 2 8 crystal structure data with residual factor R - 30% showed Cu-N to be This has recently been determined as [11,121. A similar observation has been made on transferrin where a coordinated water is lost upon freeze-drying also [131. Thus, a comparative study of solution and freeze-dried protein may prove helpful in other cases too for defining the presence of the coordinated water. The observation of Cu-0 at 2.24 f provided confirmation to the considerable spectroscopic evidence that copper in superoxide dismutase is five coordinate, the fifth coordination position being taken up by a water molecule. For the Zn site, 3 Zn-N were established at and Zn-O at We note that, given the data range, the distinction between Zn-N and Zn-0 contributions is not possible. 2.2 Oxidised vs reduced protein Blumberg et a1 [I41 used the Zn edge XANES of the native and the reduced protein to show that the near edge spectrum of the two were very similar, thus suggesting that the bridging imidazole from His-61 is coordinated to Zn in both cases. Blackburn et a1 [I 0 I extended this study to show that while the Zn-edge spectrum did not change, the Cu-edge XANES and EXAFS changed.

4 A comparison of the Cu K-edge EXAFS for the two forms showed the overall amplitude decreased by - 20% in the reduced protein, thus suggesting a decrease in the coordination number. Blackburn et a1 analysed their EXAFS data using a 3-shell model. They suggest that Cu(1) is three coordinate involving only imidazole coordination. In addition to a decrease in coordination number, they find that the Cu-N bond length is decreased to 1.94 f 0.02 A, which is again indicative of a coordination number lower than in the native protein. These results have provided the first direct structural information on the copper site of the reduced protein. 2.3 Multiple scattering effects It has been realised for some time that the outer atomic shells of an imidazole ring cause strong multiple scattering over an extended k-range in the EXAFS, and that the EXAFS can be simulated only by allowing these atcans to take a substantially shorter position Ca A under the single scattering scheme. We provide (elsewhere in the proceedings) the first quantitative examination of multiple scattering contribution from the atoms of an imidazole ring. Here, for completeness, some of the relevant results are described. I - Experiment 1 - Experiment I Figure 1. Cu K-edge EXAFS of freeze dried superoxide dismutase and a theoretical simulation based on parameters given in table 1. The multiple scattering effects from outer shell atoms of the imidazole rings have been included. The experimental spectrum has been shifted by 18 ev to match the theoretical calculation.

5 C JOURNAL DE PHYSIQUE An analysis of EXAFS data from several crystallographically well characterised compounds has been carried out to define clearly the multiple scattering effects of an imidazole ring. We find that if the multiple scattering effects are included, the thoeretical calculations can be extended to the experimental k = 0 8-I to fit the overall shape of the XANES spectrum. Figure 1 is the best simulation for the Cu K-edge EXAFS of the freeze-dried form of superoxide dismutase. This is obtained with the parameters given in table 1, which are compared with the parameters for single scattering simulations. It is clear that a rigorous analysis of the EXAFS spectrum, when multiple scattering effects are taken into account, yields correct distances of all the atoms of an imidazole ring. In addition the strong angular dependence of the multiple scattering along with the splitting of C2/C5 atoms should allow a determination of the orientation of an imidazole ring around Cu-N. Table 1. Parameters for best simulations of the Cu K-edge EXAFS spectrum of the freeze-dried powder of Cu-Zn superoxide dismutase Atoms Single scattering Multiple scattering corrected R (a) a2 (a2) R (a) Significance of structure in XANES We [21 have recently carried out detailed multiple scattering calculations to understand the XANES of a number of copper imidazole compounds which show EXAFS and XANES spectra akin to the copper site of the freeze-dried and aqueous superoxide dismutase. Figure 2 shows the experimental XANES for three imidazole complexes each with two axial ligands to the CuN4 equatorial plane. Also shown are the spectra for the Cu site of superoxide dismutase. Each of the spectra show a number of spectral features, with varying degree of resolution, suggesting significant stereochemical differences between them. We have shown that each of these features, their relative intensity and resolution are a signature of a particular aspect of the structure. For example, the peak marked 'b' arises due to the axial ligands; its intensity and position is related to the number and position of these ligands. Similarly, features marked 'd' and 'e' (and associated features ' f' and ' g', respectively) depend on the orientation of imidazole rings with respect to the CuN,, plane. 2.5 Axial ligands The first of these can be simply demonstrated by a set of single-shell calculations, fig. 3. In fig. 3a, scattering contributions with the four nitrogens at 2.01 a in a square planar arrangement are shown. Calculations are shown in which the photon polarisation is normal (n) or parallel (p) to the plane of the four nitrogen nearest neighbours; naturally the p-component of the spectrum is sensitive to scattering events within the plane while the scattering by the axial ligands

6 Energy (Hartrees) Figure 2. Experimental XANES of three copper imidaiole complexes and for the Cu site of Cu-Zn superoxide dismutase in the freeze-dried and aqueous state. 1 Hartree = 27.2 ev. shows up most in the n-component. The polarisation-averaged spectrm is shown in the right-hand panel. Figure 3b shows these contributions with two oxygen ligands included at 2.56 a. The main contribution, marked with an arrow, due to the axial ligand results in a well-defined peak in the averaged spectrum arising predominantly f ran the n-component. The position of this contribution and peak remains unchanged when the calculations are done with only one axial ligand at the same distance (fig. 3c). However, due to the weaker contribution for one axial ligand compared to two axial ligands the intensity of this peak is reduced. The effect of the position of the axial ligand is shown in fig. 3d which shows the individual n- and p-components and the average. These calculations are based on a CuN40 unit with the axial oxygen at , a distance determined from the EXAFS study of superoxide dismutase. Thus a comparison of these simple one-shell calculations allows us to establish that the peak marked 'b' in the experimental spectra arises ran the axial ligands and that the observed difference in the Cu(1mid),+(NO ) and superoxide dismutase is primarily 3 2 due to a reduction in the number of axial lxgands and its position ( figs. 3b and 3d). This is consistent with the EXAFS result of superoxide dismutase [I41 which shows that the oxygen atom is at 2.24 a compared to the oxygen atoms in C~(Imid)~ (No ) at an average distance of We note that the psition of this 3.2 feature 'b' wlll not be affected significantly when the axial ligand's distance is

7 ( JOURNAL DE PHYSIQUE increased beyond - 2.8,fi as the orbital (charge density) overlap is reduced considerably. Energy (Hartrees) Figure 3. Panels on the left hand side are the two polarisation components, p (solid curve) and n (dashed curve). The right hand panel gives the averaged spectrum. (a) One-shell calculation with Cu-N where Cu-N is at 2.01,fi; (b) with CuN402, where oxygen atoms are placed at 2.56 %'normal to the CuN4 plane. me arrowed feature corresponds to the experimental feature marked 'b', in fig. 2; (c) with CuN40, where Cu-0 is at 2.56,fi and (dl where Cu-0 is at 2.24 A. 2.6 ~elative orientation of different groups Figure 4 shows two calculations with a cluster of 22 atoms originating from the four imidazole groups and two axial oxygen atoms at 2.56 A. In (a), the two imidazole rings are perpendicular and two nearly flat with respect to the CuN4 plane. For (b), all the four imidazole rings are nearly perpendicular +a the CuN4 plane. The two calculated spectra are almost identical over the whole XANES except in the region around experimental features 'f' and 'g'. In the case of (a) both fep.tures are reproduced, whereas they merge when all the rings are perpendicular to the plane. Thus, the fact that the XANES spectra are sensitive to the relative orientation of the imidazole rings offers the exciting possibility that relative orientation of different groups around a metal site can be determined using high resolution XANES spectra.

8 Energy (Hartrees) Figure 4. Three shell calculations for CuN(Imid)402. (a) 2 imidazole rings Lr and two II to the CuN4 plane; (b) all 4 imidazole rings nearly lr. Differences in the two theoretical spectra between Hartrees are evidence for the inter-ligand scattering. 2.7 XANES spectra of the superoxide dismutase Figure 5a shows the experimental XANES spectrum of superoxide dismutase and fig. 5b-d are the various simulations using different geometrical arrangements around the Cu atom. Figure 5b is based on the crystal structure coordinates available 20 months ago in the Brookhaven Protein Databank. Figure 5d was obtained from the coordinates obtained using the EXAFS data of Cu and Zn sites in conjunction with the crystal structure data of the protein with the molecular graphics facility at Birkbeck College. Details of this work have been published elsewhere [151. Figure 5c was obtained with revised crystal structure coordinates, which show an obvious improvement. These coordinates were obtained in December 1985 from a further crystallographic refinement, R = 19% at 2 a resolution [11,12]. A comparison of figs. 5b and 5d with the experimental spectrum clearly shows that the EXAFS-generated Cu site results in a close simulation of the XANES spectrum. This opens up an exciting possibility where use is made of EXAFS and XANES for obtaining a more refined structure of a metal site in a protein, but more importantly in obtaining this information for protein in solution and also when the protein is modified either by ligand binding, change of ph or oxidation state. Figure 6 shows the sensitivity of this region where the spectra for the Cu-site of SOD in its different forms are shown. Thus, the importance of understanding the XANES region cannot be over emphasised. Acknowledgement We would like to thank the Science and Engineering Research Council for their support of this work. We are pleased to acknowledge Dr. Paul Durham for several discussions on the use of XANES. Dr. N. Binsted's contribution to this work is invaluable, as without his programme package MCURVE most of the work presented here would not have been possible.

9 CS-1136 JOURNAL DE PHYSIQUE I Energy (Hartrees) Figure 5. Experimental XANES of an oxidised solution of superoxide dismutase with a number of 3-shell calculations (see text for details). I Photon energy (ev) Figure 6. Experimental XANES of superoxide dismutase solution protein in its native, reduced and cyano-bound form. References [I] Hasnain, S.S. in synchrotron Radiation and Biophysics, ed. A. Bianconi, Springer-Verlag, Berlin (to be published). [21 Alagna, L., Strange, R.W., ~urham, P., and Hasnain, S.S., J. ~m. Chem. Soc. (submitted), also available as a Daresbury Preprint DL/SCI/P509E (May 1986). [31 Gurman, S.J., Binsted, N., and Ross, I., J. Phys. C17 (1984) 143. [41 Lee, P.A., and Pendry, J.B., Phys. Rev. (1975) [51 Blackburn, N.J., ~asnain, s.s., Diakun, G.P., Knowles, P.F., Binsted, N., and Garner, C.D., Biochem. J. 213 (1983) 765. t61 Guman, S.J., Binsted, N., and Ross, I., J. Phys. (1986) [71 Durham, P.J., Pendry, J.B., and Hodges, C.H., Comput. ~hys. commun. 25 (1982) 193. t81 Richardson, J.S., Thomas, K.A., Rubin, B.H., and Richardson, D.C., Proc. Natl. Acad. Scl. 22 (1975) [9] Valentine, J.S., and Pantoliano, M.W., in Copper Proteins, eds. T.G. Spiro (John Wiley, New York and London) Chapter 8, [lo] Blackburn, N.J., Hasnain, S.S., B~nsted, N., Diakun, G.P., Garner, C.D., and Knowles, P.F., Biochem. J. 219 (1984) 985. [11] Tainer, J.A., Getzoff, E.D., Richardson, J.S., and Richardson, D.C., Nature 306 (1983) 284. [I21 Tainer, J.A. (Personal communication, December 1985). I131 Garratt, R.C., Lindley, P.F., Evans, R., and Hasnain, S. S., these proceedings. t141 Blumberg, W.E., Peisach, J., Eisenberger, P.M., and Fee, J.A., Biochemistry 11 (1978) [I51 Blackburn, N.J., and Hasnain, S.S., in Biological and Inorganic Copper Chemistry, eds. I.D. Kavlin and J. Zubriata. (Aderin Press, New York), (1986).