Spectroscopic Methods

Circular Dichroism (CD) Spectroscopy

The selection rules for CD spectroscopy allow for differential absorption of circularly polarized light. This allows observation of bands that are not detectable using standard absorption spectroscopy; the CD signal can also be positive or negative, which can better resolve overlapping bands.  CD is further used for spectroscopic assignment as transitions are governed by the magnetic dipole operator.  This technique is particularly well suited for protein environments which are inherently chiral (a requirement for CD activity).

K. E. Loeb, T. E. Westre, T. J. Kappock, N. Mitic, E. Glasfeld, J. P. Caradonna, B. Hedman, K. O. Hodgson and E. I. Solomon.  “Spectroscopic Characterization of the Catalytically Competent Ferrous Site of the Resting, Activated and Substrate-Bound Forms of Phenylalanine Hydroxylase.” J. Am. Chem. Soc. 119 (1997): 1901.

Magnetic Circular Dichroism (MCD) Spectroscopy

In a magnetic field, the selection rules change for the absorption of circularly polarized light.  As with absorption and CD, the energies of ligand field d-d transitions observed by magnetic circular dichroism (MCD) provide insight into coordination number and geometry of the metal while charge-transfer transitions yield information on charge distribution in ligand-metal bonds.  Detailed analysis of the field- and temperature dependence of the bands can be used to extract the ground-state sublevel splittings.

M.I. Davis, A.M. Orville, F. Neese, J.M. Zaleski, J.D. Lipscomb, and Edward I. Solomon. “Spectroscopic and Electronic Structure Studies of Protocatechuate 3,4-Dioxygenase: Nature of Tyrosinate-Fe(III) Bonds and Their Contribution to Reactivity” J. Am. Chem. Soc.124(2002):602.

Electron Paramagnetic Resonance (EPR) Spectroscopy

Electron paramagnetic/spin resonance (EPR/ESR) spectroscopy identifies and quantitates the degree to which metal and ligand atomic orbitals contribute the ground state wavefunction of transition metal sites in model compounds or metalloproteins containing unpaired electrons.  Information regarding ligand-field energy splittings and the implied metal coordination geometries can also be obtained.  Variable frequency (X- and Q-band) allows one to resolve different features of the spectrum while single crystal EPR can be used to correlate the orientation of the wavefunction with the structure of a metalloenzyme active site.

A. E. Palmer, D. W. Randall, F. Xu and E. I. Solomon. “Spectroscopic Studies and Electronic Structure Description of the High Potential Type 1 Copper Site in Fungal Laccase:  Insight Into the Effect of the Axial Ligand.” J. Am. Chem. Soc. 121 (1999):7138.

Resonance Raman (rR) Spectroscopy

rR spectroscopy evaluates vibrational modes that are enhanced by laser radiation whose energy matches that of a charge transfer transition.  The fact that a specific charge transfer band is involved gives the technique a very important selectivity advantage: only molecular vibrations associated with that charge-transfer transition are observed. Isotopic shifts and rR enhancement profiles of proteins and model complexes provide further information about the ground and excited state electronic structures, vibrational modes and excited state geometries.

M. J. Henson, P. Mukherjee, David E. Root, T. D. P. Stack and E. I. Solomon. “Spectroscopic and Electronic Structural Studies of the Cu(III)2 Bis--oxo Core and its Relation to the Side-on Peroxo-bridged Dimer.” J. Am. Chem. Soc. 121 (1999): 10332.

Variable-Temperature Absorption Spectroscopy

Transitions to and from excited electronic states can provide a very detailed picture of the electronic and geometric structures of proteins and model complexes.  Our ability to compare band intensities for MCD and absorption at low temperatures gives us a powerful method of defining the nature of electronic transitions.  Band shape changes over a large temperature range provide further information about these excited states.

T. C. Brunold, D. R. Gamelin, and E. I. Solomon.  “Excited-State Exchange Coupling in Bent Mn(III)-O-Mn(III) Complexes: Dominance of the / Superexchange Pathway and Its Possible Contributions to the Reactivities of Binuclear Metalloproteins.”  J. Am Chem. Soc. 122(2000):8511.

X-ray Absorption Near-Edge Spectroscopy (XANES)

Investigations of the absorption spectrum at or near the onset of absorption of core levels (i.e. K-edges for excitation of n = 1 electrons, L-edges for n = 2 electrons, etc.) provide important information that can relate to the bonding characteristics of a molecule and the nature of the species.  Since the donor orbital is highly localized on a particular atom, selection rules are derived from atomic orbital considerations.  The localized nature of the donor orbital also allows for the quantitation of specific covalency contributions to certain bonding interactions.

T. Glaser, K. Rose, S. E. Shadle B. Hedman, K. O. Hodgson, and E. I. Solomon.  “S K-edge X-ray Absorption Studies of Tetranuclear Iron-Sulfur Clusters: -Sulfide Bonding and Its Contribution to Electron Delocalization.” J. Am. Chem. Soc. 123(2001):442.

Extended X-ray Absorption Fine Structure (EXAFS)

Beyond the onset of the absorption edge for ionization, the emitted photoelectron wave interacts with nearby atoms and creates interference effects that are visible at energies above the absorption edge.  These interference patterns can be deconvoluted to provide accurate geometric information about atoms near the photoionized atom.  Furthermore, theoretical modeling using programs such as GNXAS and FEFF can provide additional insight into structural parameters.

Serena DeBeer George, Markus Metz, Robert K. Szilagyi, Hongxin Wang, Stephen P. Cramer, Yi Lu, William B. Tolman, Britt Hedman, Keith O. Hodgson, and Edward I. Solomon.  “A Quantitative Description of the Ground State Wavefunction of CuA by X-ray Absorption Spectroscopy: Comparison to Plastocyanin and Relevance to Electron Transfer”. J. Am. Chem. Soc. 123(2001):5757.

Photoelectron Spectroscopy (PES)

Detailed bonding information can be obtained from the investigation of photoionization behavior of valence and core electrons.  Photoelectron spectroscopy (PES) provides a direct probe of energy-correlated electron density, i.e. the energy level diagram for a molecule.  Using the variable-energy synchrotron source at the Stanford Synchrotron Radiation Laboratory (SSRL), the atomic contributions to molecular orbitals can be obtained for valence molecular orbitals.  In addition, variable-energy PES is used to directly probe electronic changes that occur upon ionization.

J. B. Reitz and E. I. Solomon. “Propylene Oxidation on Copper Oxide Surfaces:  Electronic and Geometric Contributions to Reactivity and Selectivity.” J. Am. Chem. Soc. 120 (1998):11467.