Resonance Raman studies of protein-cofactor interactions in wild type and mutant purple bacterial reaction centers

The focus of this work is to characterize the interactions between the functionaly important cofactors and the protein environment around these cofactors in photosynthetic bacterial reaction centers (RCs) that are genetically modified and exhibit novel electron-transfer properties. The genetic modifications are in the vicinity of special pair and two accessory bacteriochlorophylls. These modifications introduce (1) hydrogen bonding to the accessory bacteriochlorophylls, (2) positive charge in close proximity to the accessory bacteriochlorophylls, (3) dipolar perturbations to the accessory bacteriochlorophylls, and (4) axial ligand changes to the special pair. The structural, vibronic, and electronic properties of the perturbed cofactors are related to the novel electron-transfer properties of the mutant RCs.; The major technique used in the studies is resonance Raman spectroscopy, which has the advantage of selectively probing the cofactors of interest without interference from other cofactors. Selectivity is achieved via excitation of the Qy absorption bands because these absorptions of the different cofactors are well resolved. The Raman scattering is companied by fluorescence emission from both impurities and the RC itself, so shifted-excitation Raman difference spectroscopic (SERDS) technique is used to remove the fluorescence background. The main focus of the Raman studies is on the ground state properties, which are reflected in the frequencies of the Raman bands of the perturbed cofactors. However, some effort has expended to study the electronic excited states through the intensities of the Raman bands.; A key conclusion of the studies is that single amino acid replacements (mutations) can have significant impact on the structure of the cofactors, but these structural perturbations do not necessarily have dramatic effects on RC function. Another conclusion is that it is very unlikely that there is a "magic" residue that breaks the symmetry of the two protein branches and dictates the unidirectionality of electron transfer in wild type RCs. It is more likely that the overall protein environment in each branch controls the properties of the cofactors via electrostatic effects and hence, controls the charge separation dynamics of the RC.