The cumulative knowledge on the primary events in bacterial photosynthesis is reaching a point where structure-function relationships can be examined on a molecular level. However, before real progress can be made a very significant hurdle yet exists: methodology must be developed to allow reconstitution of reaction center (RC) and light-harvesting (LH) complexes from their purified components (i.e., bacteriochlorophyll, polypeptides, etc.) Toward this end we have very recently reversibly dissociated the LH complex of Rhodospirillum rubrum (called B870) to a smaller form absorbing at 820 nm and obtained quantitative yields. We plan to extend this methodology and attempt to reversibly dissociate the 820 nm form to its component parts. If this is successful, then we will probe structure-function relationships by preparing reconstituted LH complexes in which the structure of the pgiment and Aplha- and Beta- polypeptides are systematically varied. The methodology we have found to be effective in reversibly dissociating the LH complexes will also be utilized with RC complexes. If we can successfully reversibly dissociate the RC into its fundamental components, this will be of great importance because of the special role the reaction center plays in energy transduction in photosynthesis. On a larger structural scale we will conduct experiments designed to help understand the fundamental mechanism of coupling of energy yielding reactions to energy utilizing reactions in bioenergetic membranes. The highly cooperative state of the photosynthetic units will be structurally and biochemically characterized and the control of its reversible conversion to a non-cooperative state will be explored. The methodology to be used will combine measurements of RC activity, metabolite transport, photophosphorylation, chemical cross-linking, analysis of protein components and electron microscopy. Physical properties of the RC and LH will also be compared. In addition to full separation and reconstitution of the RC components, the development of methodology to replace individual components (e.g., ubiquinone) in more intact systems will be continued. Also to be continued is our systematic characterization of oxidation and protonation state changes in ubiquinone during the primary photochemical events. The methodology to be employed involves collecting light-induced absorbance spectra as well as measuring Resonance Raman change spectra in the UV/VIS/NIR regions at low temperature.
