X-Ray Absorption Spectroscopy

This technique, which takes advantage of the high-intensity, high-collimation x-ray sources at synchrotron radiation facilities, can yield direct local structural information about a metal center in a metallobiomolecule. Detailed assignment of ligands, site symmetries, and metric parameters (metal-ligand distances) are available even for amorphous (non-crystalline) samples. Dr. Scott is an international expert in the application of this technique to metallobiomolecules. His research is conducted at the Stanford Synchrotron Radiation Laboratory and this technique is available to collaborators in the MRIL by advance arrangement [Robert Scott, UGa].

X-ray Absorption Spectroscopy, edges

Acronyms, synonyms

• X-ray Absorption Spectroscopy
• X-ray Absorption Near Edge Structure

Measured physical quantities

• X-ray induced dissociation of core electron; core- to valence-level electronic transitions

Information available

• Metal-site oxidation state
• Coordination geometry/number
• Qualitative ligand environment
• Metal-localized charge-density changes from one sample to another

Information NOT available, limitations

• Usually only relative oxidation state is available from comparisons of edges, and then only if the structure of the site in both oxidation states is constant
• If a mixture of sites (of the same metal) is present, deconvolution of spectrum is problematic
• Determination of coordination geometry and number is more difficult with metal sites of low symmetry
• Insensitive to magnetic or isotopic properties of metal sites

Examples of questions that can be answered

• Does metalloenzyme reduction or oxidation affect this metal site?
• Does a particular treatment (e.g., addition of substrate or inhibitor) affect the coordination geometry or coordination number at this metal site?
• What is the qualitative nature of the ligand donor-atom set for this metal site (e.g., mostly soft S-containing ligands or hard (N,O)-containing ligands)?

Major advantages

• Can obtain (indirect) structural information on amorphous samples (e.g., frozen solutions)
• Technique is element-selective; no interference from other metals

Major disadvantages

• Information obtained is mostly qualitative; theory of edge structure is not well understood
• Requires fairly high concentration of metal sites
• Requires synchrotron radiation beam time

Sample constraints

• Only straightforward for elements with atomic number greater than or equal to 20 (Ca)
• Metal concentration must be greater than or equal to 0.3 mM (more for lower atomic numbers)
• Sample must be quick-frozen, possibly with glassing agent (e.g., glycerol, ethylene glycol)
• Sample volume is ca. 0.1-0.2 mL

Extended X-ray Absorption Fine Structure (EXAFS)

Acronyms, synonyms

• X-ray Absorption Spectroscopy
• Extended X-ray Absorption Fine Structure
• X-ray Absorption Fine Structure

Measured physical quantities

• Scattering of x-ray induced photoelectron by electron density around neighboring atoms

Information available

• How many of what type of atom are at what distance from a metalsite
  Coordination number
  Type of coordinating atom
  Metal-ligand distance

Information NOT available, limitations

• Coordination geometry is not available; only radial distances are determined
• Structural information is limited to a radius of ca. 4-5 Å from metal site
• Coordination numbers are usually only accurate to ca. 25%
• Ligand atom type is usually only accurate to DZ ca. ± 15% (Z is atomic number)
• If a mixture of sites (of the same metal) is present, deconvolution of spectrum is impossible
• Insensitive to magnetic or isotopic properties of metal sites

Examples of questions that can be answered

• What is the ligand environment around this metal site?
• Does a particular treatment (e.g., addition of substrate or inhibitor) affect the structure at this metal site?
• Is this metal part of a multinuclear cluster?

Major advantages

• Can obtain direct structural information on amorphous samples (e.g., frozen solutions)
• Technique is element-selective; no interference from other metals
• Metal-ligand distances can be measured very accurately (± 0.02 Å or better)

Major disadvantages

• Requires high concentration of metal sites
• Requires synchrotron radiation beam time
• Coordination numbers and atom-type determinations are relatively inaccurate
• Often does not give a unique determination of ligand environment; depends on simulation and curve-fitting

Sample constraints

• Only straightforward for elements with atomic number greater than or equal to 20 (Ca)
• Metal concentration must be greater than or equal to 1 mM (more for lower atomic numbers)
• Sample must be quick-frozen, possibly with glassing agent (e.g., glycerol, ethylene glycol)
• Sample volume is ca. 0.1-0.2 mL