Resonance Raman Spectroscopy

This technique provides information about the vibrational characteristics of metal centers, leading to structural information. Using laser excitation, transition metal centers that absorb light can be selectively probed even in very large metallobiomolecules. The MRIL has available two lasers to pump a dye laser making excitation wavelengths from 350-750 nm available [Mike Johnson, UGa].

Acronyms, synonyms

• Resonance Raman
• Resonantly enhanced Raman

Measured physical quantities

• Intensity of inelastically (Raman) scattered photons as a function of frequency separation (Raman shift) from a monochromatic source

Information available

• Vibrational frequencies of the chromophore in resonance with the incident frequency
• Metal coordination geometry and ligand environment via analysis of vibrational frequencies
• Metal-ligand bond strengths via analysis of vibrational frequencies
• Electronic assignments via resonance excitation profiles (REPs)

Information NOT available, limitations

• Insensitive to magnetic properties of metal centers
• Not useful for investigating non-chromophoric metal centers, e.g. Zn²⁺
• Cannot be used for absolute quantitation of metal centers

Examples of questions that can be answered

• What is the oxidation state, spin state, and axial ligation of this heme?
• What is the effect of this inhibitor/substrate/mutation on the metal coordination sphere?
• What type of Fe-S cluster is this?
• What is the Fe-O-Fe bond angle in this diiron center?

Major advantages

• Exquisitely sensitive to minor structural changes at metal sites
• Selective determination of the vibrational properties of individual chromophores is possible in proteins with multiple chromophoric prosthetic groups
• Can monitor kinetics of changes at the metal site down to the picosecond time-scale using time-resolved techniques
• Requires miniscule amount of material for low-temperature studies
• Can be used at room temperature or low temperature with (frozen) solutions, solids, or single crystals

Major disadvantages

• Indigenous fluorescence or traces of fluorescent impurities can prevent the acquisition of RR spectra
• Meaningful vibrational analysis requires extensive isotope substitution data
• The factors controlling the extent of resonance enhancement of discrete vibrational modes are generally not well understood (i.e., some vibrations may not be observed due to negligible resonance enhancement)

Sample constraints

• Require at least 10 µL pure protein as concentrated as possible
• Must avoid laser-induced sample degradation by cooling, spinning, or flowing