Investigation of the quinone sites in reaction centers from Rhodobacter sphaeroides by pulsed EPR and spectral simulations

The reaction center from Rhodobacter sphaeroides is responsible for the primary process of photosynthesis where light is converted into a membrane potential. This is achieved by electron transfer from a special pair of bacteriochlorophylls to two acceptor quinones, QA and QB. Both quinone sites are occupied by identical ubiquinone molecules, yet take on very different roles. QA can only accept and donate a single electron, and serves as a one-electron gate to QB. QB can accept two electrons and, by means of proton-coupled electron transport, will become fully reduced into quinol. The quinol then dissociates from the reaction center to be replaced by another oxidized ubiquinone molecule. The QA- and QB- semiquinone intermediate states can both be readily trapped by photoreduction or chemical reduction methods, making them well-suited for study by electron paramagnetic resonance (EPR). In this thesis, pulsed EPR is used to explore the magnetic interactions between the semiquinones and the protein environment, the solvent, and even within the quinone molecule itself. The magnetic coupling constants that characterize these interactions are estimated from the experimental data by spectral simulations and, wherever possible, are used to obtain high-resolution structural information on the semiquinones and their environments. The hyperfine and nuclear quadrupole interactions with the nitrogen hydrogen bond donors of the semiquinones are investigated first. A multi-frequency approach, taking advantage of measurements at S-, X-, and Q-band (approximately 3.6, 9.7, and 34 GHz, respectively), allows for an accurate characterization of the principal values and directions of the magnetic tensors. These tensors provide insight into the hydrogen bond geometry, with emphasis given to how the coupling constants are related to the histidine Nδ–semiquinone hydrogen bond strength. Next, the role of the methoxy groups in establishing the redox potential difference between QA and QB is explored by site-specific 13C labeling. The dihedral angles of the methoxy groups with respect to the quinone plane are estimated by comparing the hyperfine couplings with Density Functional Theory calculations. The difference in the QA and QB 2-methoxy dihedral angles is found to contribute at least 160 mV to the quinone redox potential difference, making this substituent an essential component of interquinone electron transfer. Orientation selective Q-band ENDOR measurements are then performed on fully deuterated reaction centers, providing the Euler angles describing the locations of the hydrogen bonded protons with respect to QB-. A 10-15° rotation of the semiquinone in comparison with crystal structures is observed. An EPR-determined structure for QB- may reveal new protein interactions with the semiquinone. Finally, a full pulsed EPR characterization of reaction center mutants at chain M residue 265 and the QA-QB- biradical are presented. The concepts developed in the previous chapters are applied here to understand the effect of mutations and the presence of two semiquinones to the quinone site structure