Protein Control of Cofactor Function

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Light energy conversion in the photosynthetic reaction center (RC) involves an initial electron transfer, followed by several more steps that stabilize the charge separation. The primary and secondary electron acceptor quinones, QA and QB, in the RC of Rhodobacter sphaeroides are chemically identical (both ubiquinone-10) but exhibit very distinct properties. Thus, they provide an ideal model for understanding how proteins determine the properties of bound cofactors. This research focuses on the origins of the distinct properties of QA and QB, which determine the nature (kinetics and energetics) of the electron and proton transfer reactions between the quinones. Specific projects will study: (1) how small weak acids reactivate ("rescue") a wide variety of mutants that are impaired in proton uptake and proton transfer to QB; (2) QA site mutants at residue M265, to be further characterized by X-ray structure determination, ENDOR and pulsed EPR studies on Zn-substituted RCs, and by computational energy minimization; (3) the origin of the QA- semiquinone IR spectrum - the "anion band" - using M265 mutants which have striking and characteristically altered QA- IR spectra; (4) the role of quinone substituents in determining QA and QB function, using synthetic analogues; (5) the role of the proximal and distal QB sites in the 1st electron transfer, using mutagenesis, functional assays and FTIR; (7) the enthalpic and entropic contributions to the temperature dependence of the 2nd ET. In all projects, free energy levels will be determined by delayed fluorescence, kinetic and equilibrium consequences by absorption spectroscopy, and structural implications by FTIR, X-ray diffraction and ENDOR spectroscopy.Catalysis is absolutely essential to life, and almost all metabolic catalysis is performed by proteinaceous enzymes. However, the catalytic mechanism is truly understood for very few enzymes, if any. This is largely because of the limited observability of the underlying atomic/molecular interactions. However, the protein-ligand interactions that facilitate enzyme action on a substrate are the same as those that modify cofactor properties, such as prosthetic groups and coenzymes, and many of these are spectroscopically well-endowed. This research describes studies on the photosynthetic reaction center (RC), which has 8 spectroscopically-active cofactors in a highly symmetrical arrangement, but with strong functional asymmetry. It therefore provides an exceptional system for identifying the protein-cofactor interactions responsible for the different functionality of chemically identical cofactors. In addition to at least 8 different electron transfer reactions, the RC also performs proton transfers, substrate (quinone) binding and chemistry, and protein (cytochrome) recognition, docking and binding. The intellectual merit of this project is the chance to observe, understand and manipulate, at a microscopic level, the interactions that underlie the uniqueness of biological mechanisms. The broader impact derives from these attributes, which make this project an exceptional one for training young scientists, with a substantially multidisciplinary component. The results are expected to contribute to a deeper understanding of photosynthesis and respiration - processes that are central to global and human survival and health, and which have formed a strong basis for teaching, mentoring and motivating students in the classroom, as well as aiding and abetting high school teachers.