Photosynthetic reaction centers (RCs) are one of life's most ancient and useful devices, allowing the biosphere to exploit the abundant solar energy continuously striking our planet and to diversify into a huge number of species with distinct ways of making a living. All known RCs have symmetric structures, using two similar (or identical) membrane-inserted polypeptides to form a dimeric core, which binds the cofactors through which electrons are transferred across the membrane. This symmetric arrangement gives rise to two branches of cofactors, down which light-driven electron transfer could proceed from a 'special' pair of chlorins (chlorophyll derivatives) at the top. The first two members of each branch are chlorins, while the third is a quinone. It is known that the initial electron transfer occurs almost exclusively along one of the two branches in the well-characterized type 2 RCs. Although the origins of this strong asymmetry are still debated, much of it can be explained by the fact that the protein-provided environment of the 'inactive branch' is made less conducive for electron transfer. Previous efforts demonstrated that Photosystem I (PS1) can use both of its branches almost equally. This project is aimed at extending those results in order to understand how directionality can be influenced. The central question is: "How does nature direct electron transfer when there is more than one way for the electrons to go?" This question will be addressed by genetic manipulation of the core polypeptides in the green alga, Chlamydomonas reinhardtii. A collection of core polypeptides with mutations near the primary electron donor/acceptor pairs will be screened by time-resolved optical spectroscopy on the nanosecond timescale to identify those mutations that alter directionality. This will be followed by advanced biophysical analysis, including electron paramagnetic resonance (EPR) and ultra-fast optical spectroscopy, to determine the effect of the mutations upon the initial charge separation events. Although PS1 is currently the best system in which to explore these questions, indications are that it is possible to create a better one. The project incorporates a plan to create a system to explore the use of asymmetry in photosynthetic RCs by converting the homodimeric type 1 RC of Heliobacterium modesticaldum into a heterodimeric RC. This will allow the introduction of asymmetry in a controlled fashion (one amino acid residue at a time). Rather than merely trying to undo what evolution has wrought, the planned approaches explore ways of biasing charge separation between the two pathways, recapitulating evolution and perhaps exploring alternative mechanisms.
Broader Impact The PI's lab has a good track record of broad inclusion of undergraduate and graduate students from diverse backgrounds as team members in the scientific enterprise. They receive a broad training in interdisciplinary science at the interface of chemistry, biology, and physics. In a previous NSF award the PI was involved in the development of two new courses, which will be continued. The first is a combined lecture/lab course on the diversity of bioenergetic mechanisms, in which the underlying goal is to teach science in the same way that science is done, with a focus on exploring the scientific literature and developing presentation skills, presenting scientific data, designing and executing experiments, and finally writing and assessing proposals. The other course focuses on the relationship between evolution, religion and philosophy, and is taught in a seminar style to undergraduates with a wide range of majors. Its purpose is to provide students a safe space in which to explore topics regarding the implications of evolutionary theory to religious beliefs and philosophical/ethical positions.