The ultimate goal of this research is to achieve a unified molecular level understanding of the photochemical processes in the bacterial photosynthetic reaction center (RC). The RCs of both bacteria and plants feature two quasi-symmetric branches of potential electron carriers. In the bacterial RC, only the L-branch (also called the A branch) is utilized for trans-membrane photo-induced electron transport. In the native RC, the M-branch (also called the B branch) is inactive. Recent work from this project has delineated conditions wherein the M-branch cofactors can be induced to support full trans-membrane electron transfer, albeit in low yield. This advance has opened the door for detailed explorations of the origins of the unidirectional electron transfer in the native RC, and of electron transfer along the entire normally inactive M-branch of RC cofactors. Such studies are the basis of this research. This work will put current models for the high quantum yield of charge separation along the L-side in the native RC to the ultimate test of consistency via manipulation and study of the same processes along the normally inactive M branch. The findings will help to elucidate the mechanistic underpinnings of electron transfer in the RC as a unified whole. From a wider perspective, the proposed work will make the normally inactive M-side cofactor chain fully accessible in a robust manner for a variety of next-generation studies in many laboratories. This includes new avenues for exploring novel intermediates and conformational or protonation states associated with the QA/QB two-electron gate function that are normally not produced or formed only transiently in the normal course of L-side charge separation. One goal will be to manipulate the cofactor content and the free energies of both the L and M-side charge-separated states to give a very high (approaching quantitative) yield of electron transfer to the normally inactive M-branch. A second goal will be to probe the contribution of electronic couplings along with energetics to directionality (i.e., initial L-side versus B-side charge separation). A third goal will be to increase the rate and yield of electron transfer from the M-side bacteriopheophytin intermediary electron acceptor to the terminal quinone acceptor QB, and in doing so test current understanding of the analogous L side processes. These goals will be pursued via static and time-resolved (femtoseconds to seconds) spectroscopic studies of RC mutants to derive the rates and yields of the charge separation and recombination processes and the relative free energies of the participating states. This research is central to understanding the primary photochemical events in the bacterial RC, and will continue to provide guideposts for research being conducted on photosystems I and II from green plants.
Broader Impacts: This research will continue to have a demonstrated impact in the participation of undergraduates and underrepresented groups in research and science, and in the broad multidisciplinary training the researchers receive. The integration of research ideas into teaching and educational development will continue to be a focus of the Co-PIs, via three projects. (1) A web-based tutorial will be developed that can be tailored for undergraduate or high school students on the general topic "Why is grass green and blood red?" (2) Two undergraduate physical-chemistry laboratory experiments will be extensively upgraded to exploit the spectroscopy of chlorophyll and related chromophores, and the energy/electron transfer processes critical to photosynthesis, to teach molecular electronic spectroscopy and kinetics from application-oriented perspectives. (3) A broad, science-based lecture on general and fundamental aspects of photosynthesis with links to interests in gardening will be developed to be part of an adult education outreach program at the Missouri Botanical Garden. Despite the undeniable relevance of photosynthesis, the program has not had such a lecture/education component. Finally, in addition to the traditional means of dissemination (publications, meetings), local and national popular print and web-based publication venues will continue to be exploited through contacts with science writers in order to disseminate photosynthesis research (and contributions made under this project) to a broad spectrum of individuals.
