• What type of Fe–S cluster is this? Resonance Raman • What is the Fe–O–Fe bond angle in this diiron center? Spectroscopy Major advantages • Extremely sensitive to minor structural and electronic Roman S. Czernuszewicz & Marzena B. Zaczek changes at the active site. • University of Houston, Houston, TX, USA Selective determination of the vibrational properties of individual chromophores possible in proteins with multiple chromophoric prosthetic groups. • Can monitor kinetics of changes at the metal site down to Method Summary 1 the femtosecond timescale using time-resolved techniques. • 1 Introduction 1 Requires minuscule amount of material for low- 2 Technical Background 2 temperature studies (∼10 µl). • 3 Applications 15 Can be used at room temperature or low temperature with 4 Further Reading 33 (frozen) solutions, solids, or single crystals. 5 References 34 Major disadvantages • Fluorescence or traces of fluorescent impurities can prevent acquisition of RR spectra. • Meaningful vibrational analysis requires extensive isotope substitution data. METHOD SUMMARY • The factors controlling the extent of resonance enhance- ment of discrete vibrational modes are generally not well Acronyms, synonyms understood. • Resonance Raman (RR) • Resonance Raman effect (RRE) Sample constraints • Resonance Raman scattering (RRS) • Requiresatleast10µl of pure protein (∼1mM). Measured physical quantities • Intensity of inelastically (Raman) scattered photons (i) as 1 INTRODUCTION a function of the wavenumber separation (Raman shift) from the elastically (Rayleigh) scattered photons and Resonance Raman (RR) spectroscopy is a powerful and (ii) as a function of the exciting radiation wavelengths versatile technique for the study of both vibrational and (three-dimensional spectrum). electronic structures of chromophoric molecular systems. RR spectra are obtained by irradiation of the sample with a Information available monochromatic light source whose energy is close to that of • Vibrational frequencies of the chromophore in resonance an electric-dipole-allowed electronic absorption band. Most with incident frequency. of the Raman bands are attenuated by the absorption, but • Metal coordination geometry and ligand environment via some bands may be greatly enhanced. This effect arises from analysis of vibrational frequencies. a coupling of the electronic and vibrational transitions, and • Metal–ligand bond strengths via analysis of vibrational the vibrational modes that do show enhancement are localized frequencies. on the chromophore, that is, on the group of atoms that give • Electronic assignments via resonance excitation profiles rise to the electronic transition. (REPs). Early RR spectra1,2 indicated that only one allowed electronic transition (the resonant one) was responsible for Information NOT available, limitations the strongly enhanced intensity of the Raman scattering • Not useful for nonchromophoric metal sites. with excitation lines in the visible or near-ultraviolet region. • Insensitive to magnetic properties for metal centers, e.g., Classical examples of these are the RR spectra of such 2+ − 2− 4− Zn . inorganic species as MnO4 ,CrO4 ,TiI4,SnI4,Mo2Cl8 , • Cannot be used for absolute quantitation of metal centers. etc.1 All of the above are characterized by an enormous enhancement of the intensity of one or more totally symmetric Examples of questions that can be answered vibrational modes, together with an appearance of a long series • What are the spin state, oxidation state, and axial ligation of overtone bands (usually only one of the totally symmetric 4− 3,4 of this heme? modes). The RR spectroscopic studies of the Mo2Cl8 ion • What is the effect of this substrate/inhibitor/mutation on had verified the close relationship between the electronic this metal coordination sphere? transition and the vibrational mode to be resonance enhanced. Encyclopedia of Inorganic and Bioinorganic Chemistry, Online © 2011 John Wiley & Sons, Ltd. This article is © 2008 John Wiley & Sons, Ltd. This article was originally published in the Encyclopedia of Inorganic Chemistry in 2008 by John Wiley & Sons, Ltd. DOI: 10.1002/9781119951438.eibc0303 2 RESONANCE RAMAN SPECTROSCOPY This ion exhibits an intense absorption band at ∼525 nm, highly monochromatic beam of intense radiation, which can be which arises from the allowed δ δ∗ charge-transfer (CT) focused very precisely into a small sample under a variety of transition involving excitation of a δ-electron of the quadruple flexible sampling geometries.16–18 The sample can be moved Mo–Mo bond.5 Thus, it is not surprising that of the three rapidly through the laser beam to minimize complications totally symmetric fundamentals, ν(MoMo), ν(MoCl), and due to local heating or photochemistry. In addition to the δ(ClMoCl), it was only the ν(MoMo) stretch that showed development of laser technology, the introduction of extremely strong RR activity in the spectrum taken with excitation sensitive phototubes and later diode array transducers enabled corresponding to the δ δ∗ CT transition. Similar RR spectra Raman and RR spectra to be recorded photoelectrically rather n− 6 obtained from other metal–metal bonded M2X8 ions have than photographically, with consequent enormous saving led to the correction of their electronic spectra assignments in in recording time. Further, high-quality double and triple the visible region. monochromators, spectrographs, cutoff filters, and Fourier- The advantage of selective enhancement has quickly transform Raman interferometers of high efficiency have been made RR spectroscopy a favorite method for the study of developed,19 so that it is now possible to scan to within a few relatively large molecules such as heme proteins,7–14 whose wavenumbers of the exciting laser line and acquire excellent chromophoric center is an iron porphyrin complex (see Iron quality Raman and RR spectra of any material in any physical Porphyrin Chemistry). Excitations in the visible and near- state. ultraviolet region have produced relatively simple Raman Technical advances continue to grow, and applications will spectra, because only the vibrations associated with the heme certainly multiply. The purpose of this article is to lay out the chromophore are resonance enhanced, but the vibrations principles behind RR spectroscopy and to illustrate them with of the surrounding polypeptide chains are not. Among examples from recent research, especially in the context of those enhanced, the in-plane stretching vibrations of the bioinorganic chemistry. porphyrin ring (1000–1700 cm−1) showed the largest increase in intensity due to interactions with the π π ∗ allowed electronic transitions (400–600 nm), which are also polarized in the porphyrin plane. There was further differentiation of 2 TECHNICAL BACKGROUND this set, with bands enhanced depending upon whether the excitation wavelength was tuned to the near-UV (Soret) or 2.1 IR and Raman Basics visible bands (α and β). The RR spectra obtained by using excitation lines with λex < 500 nm, i.e., with lines approaching Molecular vibrational frequencies (10−13 –10−14 Hz) lie the energy of the Soret band, were dominated by bands in the infrared (IR) region of the electromagnetic spectrum. attributable to totally symmetric vibrations. On the other hand, Transitions to vibrationally excited states can therefore be such vibrations were not observed when λex > 500 nm were probed by direct absorption of IR photons (IR spectroscopy) used (α –β region). Instead, bands attributable to nontotally (see Vibrational Spectroscopy).20–22 All molecules except symmetric vibrations were strongly enhanced. This differential homonuclear diatomic molecules (e.g., H2,O2,N2,andthe enhancement of the Raman bands in different scattering halogens) absorb IR light. In IR spectroscopy the vibrational regimes of heme proteins is the first reported example of frequency is observed as a peak in the absorption spectrum more complex resonance behavior under multiple resonant at the absolute frequency of the absorbed IR radiation. 7–10 state conditions. It attracted the attention of theorists Alternatively, inelastic collisions of the sample molecules and inspired the development of the vibronic theory of RR with the quanta of light in the ultraviolet, visible, or near- scattering (vide infra). infrared regions can induce the same vibrational transition Progress in the field of Raman and RR spectroscopy has via an inelastic light scattering process. This Raman process been heavily dependent on laser technology. The advent of is shown diagrammatically in relation to IR absorption in accessible and relatively inexpensive laser sources in the early Figure 1. All molecules including the homonuclear diatomics 1960s has caused a revolution in Raman techniques, by largely are Raman scatterers. In Raman spectroscopy, the exciting displacing the traditional mercury discharge lamp as a Raman photon has much higher frequency and energy than the excitation source. Before lasers were available, the process molecular vibration. As a result of the inelastic collision, of obtaining a good quality Raman spectrum of anything part of the incident photon energy hν0, equal to the vibrational but the most straightforward molecular systems involved as quantum, υ υ, is retained by the vibrating molecule, while much art as science, required about 10–20 ml of sample, and the scattered photon emerges with lower frequency, ν0 − νυ,υ , was often a very time-consuming operation. While important and energy h(ν0 − νυ,υ ) (Stokes scattered