9631063 Wraight The photosynthetic reaction center (RC) provides an almost ideal system for studying the roles of internal electrostatics and conformational mobility in protein function, and this project focusses on two important topics: (1) intra-protein proton transfer, which plays a crucial role in almost all catalysis, and (2) conformational relaxations in response to internal charge generation and displacement, also of universal importance in catalytic function. Specific experiments are proposed for RCs from Rb. sphaeroides and Rps. viridis, both of which have known atomic structures. (i) Site-specific mutagenesis of selected residues in the L, M and H subunits of the bacterial RC, chosen to probe their role in proton transfer coupled to the reduction of QA and QB. Charge movements will be monitored by electrochromic effects on the RC pigments, by direct electrical measurements on membrane systems, and by infra- red spectroscopy (ii) Studies of osmotic pressure and solvent isotope effects to probe the role of internal waters as a reservoir of protons inside the protein, and of bulk water in the exchange of quinone and quinol at the QB site. Both effects will be assayed by measurements of multiple turnovers of the RC, driven at high light intensity, to reveal limiting steps in the steady-state turnover. (iii) Studies of P+ QA - charge recombination as a function of temperature and pre-illumination, using the charge recombination kinetics as an assay of the energentics, and spectral responses of the pigments as an assay of the internal electric field. The research will probe the nature of the important conformation events by controlling the RC ground state through the ionic milieu and the pH, and by using selected mutants. This work will be extented to P+ QB- recombination, over the temperature range that it works. %%% All aspects of protein structure and function depend on the myriad of electrostatic interactions between atoms and groups inside the molecule. Almost all aspects of protein function require movement of the molecule, at all levels of organization from individual atoms to coordinated motions of sections of the macromolecule. Such movements respond to and generate electrostatic effects, so that the two are intimately entwined., have been increasingly recognized in recent years. Although of very general importance, experimental systems suitable for studying these processes are extremely limited and, especially with respect to dynamic events, they are essentially restricted to light activated protein reactions. This project utilizes the photosynthetic reaction center (RC) from a bacterium as an ideal model system for studying these fundamentally important processes. The light-driven reaction of the RC generates charge movements inside the protein, which initiate molecular motions (conformational relaxations) which then modify the subsequent behavior of the charges. These events can be disentangled by selective modification of the structure of the RC by mutagenesis, and by restricting the time-evolution of the RC states by cooling to low temperatures. Conformational motions are also necessary to allow small molecules in and out of a protein, as part of the protein's functional cycle. This, too, will be studied in terms of how protons pass through the protein to reach their site of reaction, how water inside the protein is involved in this, and how water comes and goes as other molecules enter and leave their active sites. ***
