Magnetic Circular Dichroism (MCD) Spectroscopy
MCD is an optical probe of paramagnetism that details the electronic and magnetic properties of the ground states of metal centers. It can be used also to identify and assign optical transitions from metal sites. Dr. Johnson is an international expert in the use of this technique on metalloenzymes. The MRIL has an excellent facility with a temperature range of 1.5-300K and wavelength range of 180-2000 nm. Circular dichroism (CD) (at zero magnetic field) is also available using additional instrumentation. This technique is good for characterizing secondary structure in (metallo)proteins [Mike Johnson, UGa].
Circular Dichroism
Acronyms, synonyms
Measured physical quantities
- Differential electronic absorption of left and right circularly polarized light
Information available
- Electronic excited state properties and assignments (metal d-d and charge-transfer transitions and ligand p-p* transitions) for chiral chromophores or chromophores in a chiral protein environment (complementary to absorption and/or MCD studies)
- Crude estimate of protein secondary structure via UV-CD (i.e. % a-helix, b-sheet, and random coil)
Information NOT available, limitations
- Metal coordination environment can only be indirectly inferred from the observed electronic properties
- Detailed electronic assignments are difficult for low symmetry metal centers
- Difficult to relate changes in CD to specific changes in protein 3D-structure
- Insensitive to magnetic or isotope properties of metal sites
Examples of questions that can be answered
- Is this band a d-d or CT transition?
- What are the kinetics of protein conformational changes that result in change in the chirality of the protein environment or in protein secondary structure?
Major advantages
- Easy; most departments have a CD spectrometer and it is no more difficult to record a CD spectrum than an absorption spectrum
- Improved resolution of electronic transitions compared to absorption measurements
Major disadvantages
- Interpreting the signs and relative intensities of CD bands is difficult for low-symmetry chromophores
- Non-chiral chromophores (e.g., porphyrins) exhibit weak CD spectra
Sample constraints
- Sample must have an OD < 1 in the region of interest
Magnetic Circular Dichroism
Acronyms, synonyms
- Magnetic Circular Dichroism
Measured physical quantities
- Differential electronic absorption of left and right circularly polarized light in a longitudinal magnetic field
Information available
- Electronic excited state properties and assignments (metal d-d and CT transitions and ligand p-p* transitions)
- Ground state magnetic properties (g-values, spin state, zero-field splittings, magnetic couplings) via variable-temperature/variable-field studies
Information NOT available, limitations
- Metal coordination environment can only be inferred indirectly from the observed electronic and magnetic properties
- Detailed electronic assignments are difficult for low-symmetry metal centers
- Not useful for investigating non-chromophoric metal centers (e.g., Zn2+)
- Insensitive to weak magnetic interactions that are smaller than the Zeeman interaction
Examples of questions that can be answered
- What is the metal center oxidation state and spin state?
- What are the effects of inhibitors/substrate/mutations on the electronic and magnetic properties of the metal center(s)?
- What are the axial ligands on low-spin ferric heme centers?
Major advantages
- All matter exhibits MCD
- Improved resolution of electronic transitions compared to absorption measurements
- Selective determination of the electronic properties of paramagnetic metal centers via temperature-dependent studies
- Selective investigation of magnetic properties of individual metal centers via temperature and magnetic field dependence studies of discrete transitions
Major disadvantages
- Interpreting the signs and relative intensities of MCD bands is difficult for low-symmetry chromophores
- Analysis of variable-temperature/variable-field MCD data in terms of magnetic ground state properties is complicated by the need to explicitly consider transition polarization
Sample constraints
- Low temperature studies require the presence of a glassing agent (e.g. 50% (v/v) glycerol or ethylene glycol)
- Sample must have an OD < 1 in the region of interest
