Resonance Raman Experiments

Early experiments using simple alkyl cobalt compounds as models for AdoCbl fostered the notion that steric crowding around the CooC bond might be an important factor that enzymes could exploit to effect the remarkable rate enhancements for homolysis of AdoCbl (Ng et al, 1983). These studies demonstrated that the more bulky the alkyl group attached to cobalt, the faster the rate of homolysis, leading to the idea that the enzymes might distort the coenzyme and thereby weaken the CooC bond. 

Vmax for [1,1,2,2 - H4]-ethanolamine. Reprinted with permission from Harkins and Grissom, 1994, copyright 1994 American Association for the Advancement of Science. 

It has recently been possible to test this hypothesis using resonance Raman spectroscopy to measure the vibrational frequency of the CooC bond of AdoCbl while bound to the enzymes themselves. 

---

Since there are a large number of different experimental laser and detection systems that can be used for time-resolved resonance Raman experiments, we shall only focus our attention here on two common types of methods that are typically used to investigate chemical reactions. We shall first describe typical nanosecond TR spectroscopy instrumentation that can obtain spectra of intermediates from several nanoseconds to millisecond time scales by employing electronic control of the pnmp and probe laser systems to vary the time-delay between the pnmp and probe pnlses. We then describe typical ultrafast TR spectroscopy instrumentation that can be used to examine intermediates from the picosecond to several nanosecond time scales by controlling the optical path length difference between the pump and probe laser pulses. In some reaction systems, it is useful to utilize both types of laser systems to study the chemical reaction and intermediates of interest from the picosecond to the microsecond or millisecond time-scales. 

Although electron transfers in biological systems are generally expected to be non-adiabatic, it is possible for some intramolecular transfers to be close to the adiabatic limit, particularly in proteins where several redox centers are held in a very compact arrangement. This situation is found for example in cytochromes C3 of sulfate-reducing bacteria which contain four hemes in a 13 kDa molecule [10, 11], or in Escherichia coli sulfite reductase where the distance between the siroheme iron and the closest iron of a 4Fe-4S cluster is only 4.4 A [12]. It is interesting to note that a very fast intramolecular transfer rate of about 10 s was inferred from resonance Raman experiments performed in Desulfovibrio vulgaris Miyazaki cytochrome Cj [13]. 

The chelate bonding mode has been found for M(CO)4(RNSNR) (M = Cr, Mo, W)16,17 in which R is a bulky group, e.g. Bu The crystal structure determination of W(CO)4(Bu NSNBu1)19 shows an average N=S bond length of 1.58 A while the NSN angle is very acute (73.4°). The elongation of the N—S bonds with respect to the normal double bond value indicates some electron donation from the metal atoms into the tt -N=S orbital, as is also shown by resonance Raman experi-ments,16,17... 

It was assumed that hypsorhodopsin contains a deprotonated form of the Schiff base because of its blue-shifted absorption maximum. As for the geometry of the chromophore in the hypso intermediate, it was speculated that it may be trans-WYe as in bathorhodopsin [112]. However, until conclusive evidence (such as could be secured by resonance Raman experiments) becomes available, it is unclear whether hypso is protonated or not and whether isomerization of the retinal moiety is involved at the hypso stage. 

In the resonance Raman experiment, selectivity and sensitivity are achieved by the use of tunable lasers which allow the selection of frequencies in resonance with the electronic absorption of the retinal moiety. This permitted the observation of greatly enhanced scattering of the vibrational spectrum of this chromophore above the background of vibrations from the opsin matrix [130]. This technique has been found useful in the studies of a large number of biologically important molecules [131,132,229]. 

A mechanism involving deprotonation of the Schiff base nitrogen [197] as the primary event is now considered unlikely because of resonance Raman experiments and also because deprotonation would lead to blue shift rather than the observed red shift in bathorhodopsin. 

Akhtar et al. [74] proposed that, in rhodopsin, an acceptor group on the protein forms a charge-transfer complex with the unprotonated Schiff base of retinal furthermore, upon ll-cis to trans isomerization, separation of donor and acceptor moieties would occur and the Schiff base linkage would be exposed to hydrolysis. This model can now be discarded as unrealistic the resonance Raman experiments have shown that it is not an unprotonated Schiff base, but a protonated base which is bound to opsin. Further, this and related models were examined by Komatsu and Suzuki [223] using theoretical calculations, who found that charge-transfer type models cannot satisfactorily explain the red shifts seen in visual pigments. 

Figure 7. Correlation between the ethylenic (C=C) stretching frequency and the main absorption maximum of the retinyl moiety in a variety of pigments and in free retinal Schiff bases in solution. (Data, based on resonance-Raman experiments at room temperature, from refs. 221, 225, 226, and 323.)...

On the basis of these accurate calculations, it is shown that whereas the lowest singlet state can be assigned to a pure XLCT state in [Ru(I)(Me)(CO)2(Me-DAB)], its character is mainly MLCT in [Ru(Cl)(Me)(CO)2(Me-DAB)] in agreement with the time-resolved absorption/emission, IR, and resonance Raman experiments. This remarkable difference has profound consequences not only on the spectroscopy but also on the photoreactivity of this class of molecules used as sensitizers and/or catalysts in photochemical reductions. 

Nanosecond pump probc time-resolved resonance Raman experiments were carried out with two Nd YAG lasers (At=7 ns) which provide the pump- (532 nm, 4 mJ) and probe pulses (416 nm, 100 mJ), a triple polychromator equipped with an intensified photodiode array, and a slow spinning cell (25tpm). For every sample, spectra were taken for delay times of At=-20, 0, 20,100 and 500 ns, 1,10,100 and 500 is, and 1ms. ... 

When the vibronic structure in the electronic emission or absorption spectra are not well enough resolved to analyze as above, Raman data must be used in combination with the electronic spectra. The most efficient method of obtaining the distortions will be to use a pre-resonance Raman spectrum in conjunction with the electronic spectrum. (In the pre-resonance Raman experiment the exciting light is detuned just off resonance.) The pre-resonance Raman spectrum corresponds to short time dynamics and a big damping factor (Section III.F.l.c). In the short time limit, the intensities in the Raman spectrum are related to the displacements by Eq. (12). In the short time limit, the absorption spectrum becomes [42]... 

There is good agreement ( 20 mV) between potential values for surface and solution redox couples. 3) Resonance Raman experiments show the presence of expected 2,2 -bpy vibrations. 4) The visible absorption spectrum of Ru(bpy)2(vpy)2 + after electropolymerization on an optically transparent SnC>2 electrode is essentially unchanged from the spectrum of Ru(bpy)2(py)2 + in solution. 5) The films can act as ion exchangers as shown by the appearance of the Fe(CN) - ... 

MLCT state of/oc-[XRe(CO)3L2] (X - Cl, L = 4-PhCOpy X = I, L = 4-MeCOpy) is quenched by NEtj via an electron-transfer mechanism. Photolysis eventually leads to reduction of the co-ordinated ketone to the corresponding alcohol and oxidation of NEtj to Et2NH and MeCHO. As the irradiated alcohol slowly exchanges with free ketone in solution this photoreaction can be exploited to effect the reduction of ketones to alcohols using visible light. Data from resonance Raman experiments confirm that the lowest-energy absorption bands of various Re(CO)3(di-imine)X complexes are MLCT in character. " ... 

---