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X-Ray Absorption Spectroscopy of Light Elements* in Biological Systems Graham N. George Stanford Linear Accelerator Center, Stanford Synchrotron Radiation Laboratory, Stanford University, Stanford, CA 94309 (Submitted to Current Opinion in Structural Biology) *Work supported in part by the Department of Energy, Offlce of Health and Enviromental Research, and the Department of Energy under Contract DE-AC03-76SF
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. Summary
Soft X-ray absorption spectroscopy of light elements has, until recently, fallen al- most exclusively within the purview of materials science. Recent developments in techniques now allow access to this spectral range by biological scientists. This review will discuss some the recent work on X-ray absorption spectroscopy of light elements in biological systems and discuss some of the possibilities for future work. Introduction X-ray absorption spectroscopy (XAS) is a well established tool for examining the local atomic environment of heavy atoms, such as transition metal ions. XAS studies of light atoms are much less common, and are fraught with experimental difficulties which are not en- countered at higher energies. This review will examine the possibilities for X-ray absorption spectroscopy of light elements in biological systems (we will refer to elements with atomic numbers smaller than 18 (potassium) as “light” elements). This excludes the majority of X-ray absorption studies on metalloenzymes, including the recent studies of first and second transition element L-edges [l-3] which fall in the same X-ray energy range as the spectra from light ele- ments. There are excellent reviews of XAS of heavy elements in biological systems e.g. [4]. A typical X-ray absorption spectrum is shown in Fig. 1, illustrating the main fea- tures of the spectrum. The spectrum can be divided into two distinct spectral regions; the edge spectrum, and the extended X-ray absorption fine structure (EXAFS) spectrum. The edge spec- trum (which is sometimes referred to as a pre-edge spectrum or near-edge structure) is primarily comprised of dipole-allowed transitions to bound states, plus continuum resonances at some- what higher energies. For the K-edges the bound state resonances are primarily dipole allowed Is+p transitions, and the edge spectra are thus highly sensitive to electronic structure [e.g. 51. Unfortunately edge spectra are often difficult to interpret in a quantitative manner. In contrast, EXAFS is relatively simple to interpret, and can give structural information upon the first shell _ coordination with accurate interatomic distances, together with estimates of the type (atomic number) and number of the surrounding atoms. ExpPCnental Aspects For X-ray spectroscopy, the availability of synchrotron radiation provides an ideal source of X-rays. The energies of the absorption edges of some light elements are listed in table
. Carbon and Nitrogen XAS
The utility of the K-edges of carbon, nitrogen and oxygen is severely hampered by the fact that most biological molecules contain many instances of these elements. In most cases the large number of overlapping spectra would be expected to be impossible to resolve. Despite these difficulties, Mitra-Kirtly et al. [6,7] have recently showed the utility of nitrogen K-edge XAS for investigating biological molecules. In particular, these workers demonstrate that the intense low-energy n* resonance associated with the herterocyclic ring of nucleic acids pro- vides a potential method for separating nucleic acid from protein in X-ray microscopy [7]. More recently Ade et al. [8] have observed similar, but rather less pronounced, chemical shifts be- tween protein and nucleic acid at the carbon K-edge. In a particularly elegant study, these work- ers succesfully used the carbon K-edge chemical shifts to image the DNA in dried chromoso- mal material from the bean Vicia faba at a resolution of 55nm. As pointed out by Mira-Kirtly et al. [7] one promising aspect for X-ray microspectroscopy is afforded by the characteristic nitro- gen K-edge chemical shifts observed for the different nucleotide bases which might allow sen- sitivity to nucleic acid composition. Magnesium and Aluminium XAS Magnesium is thought to be an essential element for all organisms. In biological systems magnesium is always present as the Mg2+ cation, and is known to play very important roles in diverse biological processes. It is important in cellular metabolism, especially in ligat- ing the phosphate groups of adenosine triphosphate (ATP), as the so-called MgATP complex. In most cases ATP is biologically active only as the magnesium complex (or sometimes in vitro as a complex with an alternative divalent metal ion such as Mn2+ ). Magnesium also has roles in biomineralization [9] and (at least) occasionally as its ATP complex [lo]. A good example of the essentiality of Mg2+ (^) for ATP utilizing systems is provided by the kinases, these are a very widespread group of enzymes which transfer phosphate from ATP to phosphorylate aliphatic _ -OH groups. They function in diverse roles, from participation in major metabolism to cascade switches [l l] and are inactive in the absence of Mg2+, (^) but the precise role that magnesium plays incatalysisi remains uncertain. Magnesium is also well known for its role in photosynthe- sis; the magnesium porpyrin chlorophyll being the primary photoreceptor in photosynthesis of green plants. Despite it’s widespread importance, very little is known concerning the coordina- tion chemistry of magnesium in biological systems. This is mostly due to the fact that magne-
sium.is a spectroscopically silent element in that, until very recently, there were no there are no useful and easily available spectroscopic probes of the element. The recent availability of YB,, X-ray monochromator crystals [12] has now made magnesium XAS a readily available probe of magnesium coordination. Fig. 1 shows the K-edge XAS spectrum of a sample of MgO obtained by using YBh6 monochromator crystals Fig, 2 A shows the magnesium K-edge spectra of three different octahedral coordinations of the Mg2+ cation, clearly illustrating the sensitivity of the edge spectrum to the coordination environment of magnesium. Magnesium is known to have a considerable coordination chemistry [ 131 and it can be anticipated that the availability of a sen- sitive probe of coordination will provide much insight into the role of this metal in biolgy. Unlike magnesium, aluminium has no known biological function. However alumin- ium is certainly very important in biology. Toxic levels of A13+ can cause dialysis encephalopa- thy in humans, and the metal has been implicated with other degenerative disorders such as the senile dementia known as Alzheimer’s disease ( but see [14] ). Recent evidence suggests that serum transferrin from people afflicted with Alzheimer’s disease may be deficient in it’s ability to bind A13’. Aluminium has also been implicated in renal disease and pancreas acinar pathol- ogy.- Additionally, aluminium toxicity is strongly implicated in the toxic effects produced by acid rain [15]. Despite it’s biological importance very few studies of aluminium chemistry in biological systems have been published. The primary reason for this is that aluminium, like magnesium, can be regarded as a “spectroscopically silent” element in that there are few con- venient spectroscopic probes. 27Al nuclear magnetic resonance (NMR) spectroscopy can be used as a powerful probe of the coordination environment of the metal. X-ray absorption spec- troscopy should provide a powerful tool that is complimentary to 27Al NMR in providing struc- tural information on alummium in biological systems. Phosphorus and Sulphur XAS Phosphorus is very widespread and important in biology. 31P nuclear magnetic _ resonance spectroscopy provides a highly sensitive probe of the chemical nature of phosphorus, and it might be argued that phosphorus XAS provides no advantages. Because of this, and be- cause of experimental.. difficulties in obtaining monochromatic light at the phosphorus K-edge, there-&e very few reports of phosphorus K-edge XAS of biological systems. Phosphorus K- edge XAS of a range of different, compounds, have been investigated using a Ge(ll1) monochromator by George et al [16]. Subtle shifts in the position of the major peak of the
reflect the differing modes of metabolism of the three organisms. Analysis of sulphur K-edge XAS provides a direct, non-invasive, probe which can be exploited to determine the metabolic fates of sulphur in whole cells. Conclusion In summary, while it is true that X-ray absorption spectroscopy of light elements in biological systems has, to date, been very little used, we can anticipate that recent technological developments will facilitate studies not only at the sulphur K-edge, but also at the magnesium and aluminium K-edges. The chemical sensitivity of XAS, possibly combined with applications in microscopy, is expected to prove an important tool not only in the investigation of purified proteins, but also in intact specimens.
References and recommended reading.
- Cramer SP, de Groot FMF, Ma Y, Chen CT, Sette F, Kipke CA, Eichom DM, Chan MK, Armstrong WH, Libby E, Christou G, Cobley UT, Brooker S, McKee -V, Mullins OC, Fuggle JC. Ligand Field Strengths and Oxidation States from Manganese L-Edge Spectroscopy J. Amer. Chem. Sot. 1991,113:7937-7940.
- George SJ, Lowery MD, Solomon EI, Cramer SP Copper L-edge Spectral Stud- ies: A Direct Experimental Probe of the Ground-State Covalency in the Blue Copper Site in Plastocyanin J. Amer. Chem. Sot. 1993,115:2968-2969.
- George GN, Cleland WE, Enemark JH, Smith BE, Kipke CA, Roberts SA, Cramer SP, L-edge Spectroscopy of Molybdenum Compounds and Enzymes J. Amer. Chem. Sot. 1990,112:2541-2548.
- Cramer SP, Biochemical Application of X-ray Absorption Spectroscopy, in X- ray Absorption Principles, Applications, Techniques of EXAFS, SEXAFS and XANES. (ed. Konisberger DC, and Prins R) John Wiley and Sons, 1988, pp 257-
- Stohr J, NEXAFS Spectroscopy Springer-Verlag, 1992.
- -Y (^) Mitra-Kirtly S, Mullins OC, van Elp J, George SJ, Chen J, Cramer SP Determina- tion of the Nitrogen Chemical Structures in Petroleum Asphaltenes Using XANES Spectroscopy. J. Amer. Chem. Sot. 1993,115:252-258.
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Mitra-Kirtly S, Mullins OC, Chen J, van Elp J, George SJ, Chen CT, O’Halloran T, Cramer SP Nitrogen chemical structure in DNA and related molecules by X- ray absorption spectroscopy. Biochim. Biophys. Acta 1992,1132:249-254. Ade H, Zhang X, Cameron S, Costello C, Kirz J, Williams S Chemical Contrast in X-ray Microscopy and Spatially Resolved XANES Spectroscopy of Organic Specimens Science 1992,258:972-975. Lowenztam HA, Wiener S, On Biomineralization Oxford University Press, 1989. Becker GL, Chen CH, Greenwalt JW, Lehninger AL Calcium phosphate granu- als in the hepatopancreas of the Blue Crab, Callinectes supidus. J. Cell Biol. 1974,61:316-326. Stryer L Biochemistry, Third edition 1988, W.H.Freeman and Co., New York Rowen M, Rek ZU, Wong J, Tanaka T, George GN, Pickering IJ, Via GW First XAFS with a YB66 monochromator. Synchrotron Radiation News, 1993 6:25- _-Fenton D, Comprehensive Coordination Chemistry Vol 3, Chapter 23, Pre- gamon Press, Oxford. 1982 Landsberg JP, McDonald B, Watt F Absence of aluminium in neuritic plaque cores in Alzheimer’s disease Nature 1992 360:65- CIBA Foundation Symposium 169; Aluminium in Biology and Medicine, Wiley 1992 George GN, Pickering IP, Via GH, Sansone M, Prince R. Phosphorus K-edge X- ray absorption spectroscopy as a probe of chemical type manuscript in prepara- tion. George GN, Gorbaty ML Sulphur K-edge X-ray Absorption Spectrscopy of Pe- troleum Asphaltenes and Model Compounds J. Amer. Chem. Sot. 1989 111:3182- 4 .- Frank P, Hedman^ B, Carlson RMK,^ Tyson^ T, Roe AL,^ Hodgson^ KO^ A^ Large^ Re- sevoir of Sulfate and Sulfonate Residues within Plasma Cells from Ascidia ceratodes, Revealed by X-ray Absorption Near-Edge Structure Spectroscopy, Biochemistry 1987 26:4975-4979.
Fig. $ Sulphur K-edge X-ray absorption spectra of whole cells of Rhodospirillum rubrum, Rhodobacter capsulatus and the marine archaebacterium Beggiatoa. Acknowledgements I thank Dr. Britt Hedman and Professor Keith Hodgson for allowing me access to material prior to publication, and my collaborators Dr Ingrid J. Pickering and Dr Roger C. Prince for helpful discussions. The Stanford Synchrotron Radiation Laboratory is operated by the Department of Energy, Office of Basic Energy Sciences. Support for research by SSRL staff is provided by that Office’s Division of Material Science. The SSRL Biotechnology Program is supported by the NIH, Biomedical Resource Technology Program, Division of Research Re- sources. Further support is provided by the Department of Energy, Office of Health and Envi- ronmental Research.
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Energy (eV) Figure 1
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RhodospirillumRhodospirillum rubrumrubrum
RhodobacterRhodobacter capsulatacapsulata
Beggiatoa
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I (^) I I I (^2470 2480 2490 2500 ) Energy (eV)
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Figure 3
