THE APPLICATION OF X-RAY ABSORPTION SPECTROSCOPY AND MICROPROBE X-RAY TECHNIQUES TO MOLECULAR BIOLOGY
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1 THE APPLICATION OF X-RAY ABSORPTION SPECTROSCOPY AND MICROPROBE X-RAY TECHNIQUES TO MOLECULAR BIOLOGY In the same way that protein crystallography and NMR spectroscopy took a couple of decades to emerge from original 'sporting techniques' to be widely applied and reliable techniques for structural molecular biology, X- ray absorption spectroscopy (XAS) has reached a similar stage of maturity. None of these techniques is stagnant; their power is continuously growing, as improvements in data collection, the sensitivity of instrumentation and theory and computation extend the range and speed of their application. XAS-derived structural information differs from those obtained from protein crystallography and NMR spectroscopy in that it only provides such information for that part of the biomolecule within ~5 Å of the atom absorbing the X-rays and is normally more precise and accurate than those obtained by protein crystallography or NMR spectroscopy. This article describes the use of X-ray absorption spectroscopy for characterisation of metalloproteins and the interrogation of intracellular biochemistry. The X-ray Absorption Process and Structural Techniques in Molecular Biology In order to understand the type of structural information that is available from the use of XAS and synchrotronradiation-induced X-ray emission (SRIXE), it is first necessary to understand the X-ray absorption process (1,2), which involves the excitation of core electrons (Fig. 1). If the core electron is excited to an empty or partially filled orbital of the absorbing atom, it results in an absorption spectrum called the X-ray absorption near-edge structure (XANES) (3,4). The intensities of the peaks in XANES have the same symmetry and spin selection rules that apply to the more familiar UV/Vis absorption spectra. Extended X- ray absorption fine structure (EXAFS) results from the ejection of the core electron from the absorbing atom to form a photoelectron, which can interact constructively or destructively with the backscattering of the photoelectron from adjacent atoms (Fig. 1) (5). If the photoelectron interacts with more than one atom before returning to the absorbing atom, it is called a multiple-scattering (MS) process. XANES and EXAFS can be recorded by measuring the absorption of the X-rays by solid or solution samples that contain relatively high concentrations of the absorbing atom. However, the direct collection of Special Technical Feature Peter Lay Centre for Structural Biology and Structural Chemistry & Centre of Heavy Metals Research, School of Chemistry, University of Sydney, NSW 2006 absorption spectra is not applicable for most studies of interest to molecular biology, because the relatively high background absorption of the other atoms in the sample means there is very little signal on a large and sloping background. Instead, fluorescence X-rays that result from valence electrons falling back into the hole created by the X-ray absorption process are measured (Fig. 1). Since the energies of these fluorescent X-rays are specific for a given element, then most of the background fluorescence from other elements can be removed by windowing the energy range of the detector, which can lead to highly sensitive detection and structural determinations on proteins at µmmm concentrations. While XANES and EXAFS data are obtained by integrating the element-specific fluorescence peak (Kα for K-edge spectra, etc.) as the energy is scanned to produce spectral data, SRIXE experiments involve the separate integration of a range of different elemental fluorescence peaks over a short period of time and scanning across a sample to produce elemental maps. The high sensitivity of fluorescence detection enables simultaneous mapping of trace elements in cells and tissues or even thin sections of these samples at spatial resolutions that are now down to less than 100 nm, i.e., enabling the interrogation of organelles within cells. The separation of the XANES and EXAFS regions is somewhat arbitrary since the energy range of the XANES and EXAFS phenomena overlap near the absorption edge. The term X-ray absorption fine structure (XAFS) is used to describe the spectrum that includes EXAFS oscillations at low k values (energies near the absorption edge) as well as the extended region. Just as electronic absorption spectra involving valence orbitals (UV/Vis visible spectroscopy) are sensitive to the metal oxidation state, the nature of the ligands, and the coordination number, so too are XANES spectra sensitive to these factors. Analysis of this region of the XAS can provide structural information, as well as information on the degree of covalency within the metalligand bonds (3,4). However, while there have been considerable advances in computational modeling of XANES, accurate computational methods for determining detailed structural information are still being developed (4) and the number of cases in which reliable bond-length information has been obtained from the computational analysis of XANES is relatively small. Acronyms used: EXAFS extended X-ray absorption fine structure; MS multiple-scattering; SRIXE synchrotron-radiation-induced X-ray emission; XAFS X-ray absorption fine structure; XANES X-ray absorption near-edge structure; XAS X-ray absorption spectroscopy; XRD X-ray diffraction. Vol 36 No 1 April 2005 AUSTRALIAN BIOCHEMIST Page 63
2 XANES is more commonly used empirically to obtain information about the structural type of an active centre in a metalloprotein. This is normally achieved by comparing the XANES of an unknown metal centre with those of well-characterised model complexes and metalloproteins. When this is used in conjunction with modeling of the XAFS region of the spectrum, reliable structural information can be obtained on an unknown metal centre of a metalloprotein, often with a high degree of confidence. MS analysis of XAFS data, particularly when used in conjunction with XANES and other spectroscopic and structural information, is a very powerful technique for determining the threedimensional structures of species for which protein crystallographic and/or NMR spectroscopic structures are not available. This situation may apply as a result of the instability of the complex/protein and/or the requirement to study the structure in the solution phase. One of the major advantages of MS analysis of XAFS data is the ability to obtain accurate and precise structural information from any medium, so long as only one or two different coordination environments are present in the sample for a given absorbing element. The major disadvantages are that XAFS analyses do not provide an absolute determination of a structure (2) and the technique is only applicable to ~40% of proteins that are metalloproteins, however, where it is applicable, it can often provide definitive evidence for the presence of one structural type over another, as well as provide accurate and precise bond length and, in many cases, bond angle information. Even if XRD- and/or NMRderived structures of metalloproteins are known, MS analysis of XAFS data obtained from frozen solutions is extremely valuable. The relevant merits of protein crystallography and XAFS analyses and combinations of these techniques in studying metalloproteins are given in recent reviews (2,6-8). As outlined elsewhere (2,3), examples of why it is important to use XAFS, even when the structure has been determined by protein crystallography or NMR spectroscopy include (2): (i) XAFS-derived metal-ligand bond length determinations are often an order of magnitude more accurate and precise ( Å); (ii) more biologically relevant active sites are sometimes obtained than in protein crystallography when non-biologically relevant ligands become coordinated in the crystallisation process, or when non-equilibrium coordination geometries are produced as a result of the diffusion of a substrate into a preformed crystal; and (iii) minimisation of structurechanging photoreduction of the metal centers of oxidized (Fe(III), Cu(II), Mo(VI), etc.) metalloproteins, when highly focused synchrotron X-ray beams are used for protein crystallography. As a consequence, MS analysis of XAFS data should be considered as a complementary technique that, in many cases, can provide additional and more precise and accurate bond length information about the active site of a metalloprotein than does protein crystallography or NMR spectroscopy. By contrast, the latter techniques provide detailed structural information on the whole protein that is not obtainable from the analysis of XAFS data (2). Photoelectron, EXAF S continuum XANES X-ray hυ Absorbance (arbitrary units) 2p 3/2 2p 1/2 2s 1s 1.2 XANES XAFS Emitted wave Scattered wave Energy (ev) e A R as S Auger electron X-ray fluorescent photon hυ Fig. 1. The X-ray absorption process and the original of XANES and XAFS and a typical K-edge Ni XAS of a Ni-peptide complex. Adapted from reference 5. Since XAS techniques, and the related XRF (X-ray fluorescence) technique, SRIXE, can be used to obtain structures and elemental maps on any materials, they are also applicable to proteins that are membrane-bound or glycoproteins, which are difficult to analyse by other techniques, without cleaving off lipophilic or sugar residues. In addition, the use of snap-frozen solutions enables the determination of the structures of reactive sites of metalloproteins that have half-lives of less than a minute at room temperature, which are clearly inaccessible by protein crystallography or NMR spectroscopy. Another advantage is that the same degrees of precision and accuracy in the structural determination of metal centres are obtained irrespective of the size of the metalloprotein, whereas such information is rapidly attenuated with the size of the protein in protein crystallography and NMR spectroscopy. Further, the advent of synchrotron X-ray microprobes and nanoprobes with a spatial resolution of < 100 nm allows SRIXE elemental maps and XANES data to be obtained from cells, tissues and even micron-thick thin sections. This technology is providing unprecedented data on the distribution, functions, and structures of metalloproteins and other metal-containing Page 64 AUSTRALIAN BIOCHEMIST Vol 36 No 1 April 2005 λ
3 species in situations that are much closer to those which apply in vivo. Finally, XAS can be used to determine structures of active sites of intracellular metalloproteins from measurements on bulk tissues or cells when metalloproteins are induced or expressed, such that one species dominates the cellular content for a particular element. Some examples of the applications of the techniques are given below. XAS Spectroscopy on Heme Proteins Because heme proteins have many and varied roles in biology, as carriers of small molecules, switching agents, enzymes and electron transfer centres, they have been studied extensively by protein crystallography, NMR spectroscopy, and by XAS techniques (1-8). The reason for the many studies on heme proteins is not only because of their importance in molecular biology, but also because the XANES can provide diagnostic information on structures and MS analysis of XAFS effects incorporates a strong angular dependence of the MS backscattering, which is strongest when atoms are collinear, and drops off rapidly below 140º (1,2). This sensitivity enables the determination of Fe-X-Y bond angles in heme proteins where XY is a diatomic such as NO -, NO, NO +, O 2, CO, etc. Such studies have been an area of intensive investigation (both experimentally and theoretically) and one where there has been much dispute on the details of the coordination geometries. As an example, the XANES of myoglobin (Mb) is very sensitive to and diagnostic of the nature of the diatomic that is bound to the sixth site in the metal centre (e.g., HNO (MbHNO), NO (Mb II NO) or NO+ (Mb III NO)) (9, 10). It can also detect the breaking of the proximal Fe-His bond, as in the NO adduct of indolamine 2,3-dioxygenase (11). The XAFS-determined bond angles and bond lengths change dramatically in the Mb protein, which, together with changes in the effective Fe oxidation state, leads to the large sensitivity in the XANES (Fig. 2). We are now using a combination of SRIXE and micro-xanes to determine the intracellular distribution and the structures of heme proteins that are induced in different parts of cells under various stimuli. Normalised Absorbance MbHNO MbIINO MbIIINO Energy, ev 1.24 Å 1.82 Å 131 o 2.00 Å 2.09 Å MbHNO 1.12 Å 1.76 Å 150 o 1.99 Å 1.13 Å 1.68 Å 180 o 2.02 Å 2.05 Å 2.04 Å Mb II NO Mb III NO Fig. 2. The Fe K-edge XANES of MbHNO, Mb II NO and Mb III NO and the XAFS-derived structures of the active sites of the metalloproteins. Adapted from references 9 and 10. Vol 36 No 1 April 2005 AUSTRALIAN BIOCHEMIST Page 65
4 P 40 b Control 20 min 240 min a S Ca Cr Zn FT Amplitude Normalised Absorbance R ' (Å) hr 4 hr c bulk cells 4 hr 20 min control Energy (ev) Fig. 3. a) SRIXE Elemental distribution maps of single A549 human lung carcinoma cells treated with Cr(VI) (100 µm) in comparison with untreated cells. b) The Fourier transform of the Cr K-edge XAFS data reveals Cr-Cr distances (indicated by an arrow) that are typical for polynuclear Cr species with two carboxylato and one hydroxo groups as bridging ligands. c) The micro-xanes at hotspots in the cells are also consistent with the structure deduced from the XAFS of bulk cells. Adapted from reference 18. XAS and SRIXE Studies on Chromium Genotoxicity Chromium(VI) is the most commonly encountered industrial carcinogen and causes cancer of the respiratory tract (12,13). The uptake-reduction model (14) has been used to explain the genotoxicity of chromate. In this model, soluble forms of [CrO 4 ] 2- are taken up by SO 4 2- or HPO 4 2- channels and insoluble forms are taken up by phagocytosis (12-14). The intracellular reduction of chromate to chromium(iii) leads to reactive Cr(VI), Cr(V), Cr(IV) and Cr(III) complexes, as well as organic and inorganic radicals en route to the final Cr(III) products (12,13). These intermediates are believed to be responsible for the observed genotoxicity (12,13). Studies on chromate-induced genotoxicity are also important because we have shown recently that redox recycling of Cr occurs as a response to normal enzymatic oxidation processes and that carcinogenic chromate generated from such processes is likely to be responsible for the anti-diabetic and fat metabolism properties of widely used Cr dietary supplements (15,16). There is a large body of evidence that the reactive Cr intermediates are responsible for Cr genotoxicity (12,13), since they cause all of the types of genetic damage that is observed when cells, animals or humans are exposed to chromate (12,13,16). Hence XAS has been used extensively by our group to determine the structures of reactive Cr(VI), Cr(V), Cr(IV) and Cr(III) intermediates (some with a half-life of < 1 min at room temperature) with biomolecules and model complexes (2, 12, 13). In order to investigate further these intracellular processes, micro-srixe, on mammalian lung cells (and thin sections of cells) were performed in conjunction with micro-xanes on individual cells, and XANES and XAFS studies on bulk cells and macromolecules exposed to Cr(VI) and Cr(III) (17,18). After a 20 minute treatment of A549 human lung adenocarcinoma epithelial cells with 100 µm chromate, Cr was confined to a small area of the cytoplasm and strongly co-localized with S, Cl, K, and Ca (Fig. 3). After the 4 hour treatment, Cr was distributed throughout the cell, with higher concentrations in the nucleus and the cytoplasmic membrane. This timedependence corresponded to ~100% or 0% clonogenic survival of the cells following the 20 minute or 4 hour treatments, respectively, and was explained by a new Page 66 AUSTRALIAN BIOCHEMIST Vol 36 No 1 April 2005
5 cellular protective mechanism (18). Such processes may also be important in reducing the potential hazards of Cr(VI) that is proposed to be generated from Cr(III) dietary supplements in vivo (15). The predominance of Cr(III) after such treatments was confirmed by micro- XANES spectroscopy on intracellular Cr hotspots (Fig. 3). X-ray absorption spectroscopy (XANES and EXAFS, using freeze-dried cells after the 0-4 hour treatments) showed that the Cr(III) products of the intracellular reductions were polynuclear in nature from the observation of adjacent Cr atoms in the EXAFS (Fig. 3) (probably with a combination of carboxylato- and hydroxo-bridging groups and O-donor atoms of small peptides or proteins). References 1. Penner-Hahn, J. (2005) in Comprehensive Coordination Chemistry II, From Biology to Nanotechnology (McCleverty, J.A., and Meyer, T.J., eds) Vol 2, Elsevier 2. Levina, A., Armstrong, R.S., and Lay, P.A. (2005) Coord. Chem. Rev. 249, Solomon, E.I., Hedman, B., Hodgson, K.O., Dey, A., and Szilagyi, R.K. (2005) Coord. Chem. Rev. 249, Rehr, J.J., and Ankudinov, A.L. (2005) Coord. Chem. Rev. 249, Barnard, P.J. (2002), PhD Thesis, University of Sydney 6. Hasnain, S.S., and Hodgson, K.O. (1999) J. Synchrotron Radiat. 6, Hasnain, S.S., and Strange, R.W. (2003) J. Synchrotron Radiat. 10, Strange, R.W., Ellis, M., and Hasnain, S.S. (2005) Coord. Chem. Rev. 249, Rich, A.M., Armstrong, R.S., Ellis, P.J., and Lay, P.A. (1998) J. Am. Chem. Soc. 120, Immoos, C.E., Sulc, F., Farmer, P.J., Czarnecki, K., Bocian, D.F., Levina, A., Aitken, J.B., Armstrong, R.S., and Lay, P.A. (2005) J. Am. Chem. Soc. 127, Aitken, J.B., Thomas, S.E., Stocker, R., Thomas, S.R., Takikawa, O., Armstrong, R.S., and Lay, P.A. (2004) Biochemistry 43, Codd, R., Dillon, C.T., Levina A., and Lay, P.A. (2001) Coord. Chem. Rev , Levina, A., Codd, R., Dillon, C.T., and Lay, P.A. (2003) Prog. Inorg. Chem. 51, Connett, P.H., and Wetterhahn, K.E. (1983) Struct. Bonding (Berlin) 54, Mulyani, I., Levina, A., and Lay, P.A. (2004) Angew. Chem., Int. Ed. 43, Levina, A., and Lay, P.A. (2005) Coord. Chem. Rev. 249, Dillon, C.T., Lay, P.A., Kennedy, B.J., Stampfl, A.P.J., Cai, Z., Ilinski, P., Rodrigues, W., Legnini, D.G., Lai, B., and Maser, J. (2002) J. Biol. Inorg. Chem. 7, Harris, H.H., Levina, A., Dillon, C.T., Mulyani, I., Lai, B., Cai, Z., and Lay, P.A. (2005) J. Biol. Inorg. Chem., in press Vol 36 No 1 April 2005 AUSTRALIAN BIOCHEMIST Page 67
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