Heme proteins resonance Raman spectra

Mutation of His 132 to an alanine, glycine, or serine in the truncated human heme oxygenase, in accord with identification of His25 as the proximal ligand, does not alter the cpe-His band in the resonance Raman spectrum of the Fe deoxy protein (82). The mutations result in the formation of two separate fractions when the protein is expressed in... 

B2y, and >42 vibrations are expected to be polarized (p), depolarized (dp), and inversely polarized (tp ), respectively. These polarization properties, together with their vibrational frequencies, were used by Spiro and his coworkers to make complete assignments of vibrational spectra of the Fe-porphin skeletons of a series of heme proteins. They showed that the resonance Raman spectrum may be used to predict the oxidation and spin states of the Fe atom in heme proteins. For example, the Fe atom in oxyhemoglobin has been shown to be low-spin Fc(IIl). It should be noted that the A2y mode, which is normally Raman inactive, is observed under the resonance condition. Although the modes are rather weak in Fig. I-19, these vibrations are enhanced markedly and exclusively by the excitation near the B band since the A-term resonance is predominant under such condition. The majority of compounds studied thus far exhibit the A-term rather than the l -term resonance. A more complete study of resonance Raman spectra involves the observation of excitation profiles (Raman intensity plotted as a function of the exciting frequency for each mode), and the simulation of observed excitation proliles based on various theories of resonance Raman spectroscopy. ... 

Fig. 13. CO isotope effects on the resonance Raman spectrum of a heme protein with a CO molecule bound to the porphyrin Fe atom. From [32].

The heme moiety provides de novo designed heme proteins with an intrinsic and spectroscopically rich probe. The interaction of the amide bonds of the peptide or protein with the heme macrocycle provides for an induced circular dichroism spectrum indicative of protein-cofactor interactions. The strong optical properties of the heme macrocycle also make it suitable for resonance Raman spectroscopy. Aside from the heme macrocycle, the encapsulated metal ion itself provides a spectroscopic probe into its electronic structure via EPR spectroscopy and electrochemistry. These spectroscopic and electrochemical tools provide a strong quantitative base for the detailed evaluation of the relative successes of de novo heme proteins. 

Resonance Raman spectroscopy is particularly suited to the study of biological macromolecules such as heme proteins because only a dilute solution (biological condition) is needed to observe the spectrum and only vibrations localized within the chromophoric group are enhanced when the exciting frequency approaches that of the relevant chromophore. This selectivity is highly important in studying the theoretical relationship between the electronic transition and the vibrations to be resonance-enhanced. 

The enhanced signal-to-noise ratio that is provided by resonance enhancement as well as the reduced complexity of the vibrational spectrum make it possible to perform a wide variety of time-resolved studies to determine the structure of the chromophore in the photocycle intermediates. These approaches are discussed in more detail elsewhere in this volume by Kincaid with emphasis on time-resolved Raman studies of heme proteins. Room-temperature flow methods have been extensively used to obtain time-resolved spectra with time resolution ranging from seconds to microseconds.The basic idea is to flow the sample and then introduce an optical pump beam upstream from the probe to initiate the photochemical cycle. Such experiments have been performed on the millisecond and microsecond time scales. For experiments with time resolution faster than microseconds, it is necessary to convert the setup to a two-pulse, pump-probe technique where the time resolution is established by the delay between the pump and probe laser pulses. The time resolution of this approach can be increased to around 1 psec beyond this point increased time resolution will be achieved only with reduced spectral resolution according to the uncertainty principle. 

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