Primary Electron Transfer Processes in Photosynthetic Bacterial Reaction Centers

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HoltenMCB 0077187The bacterial RC is a highly symmetric protein that has two chains of pigments, referred to as the L and M branches that are essentially mirror images of one another. However, charge separation takes place exclusively via the bacteriochlorophyll, bacteriopheophytin, and quinone cofactors on the L branch (BL, HL, and QA, respectively). The role of the M branch pigments (BM, HM, and QB-), or more specifically how "unidirectionality" of electron transfer to the L branch is achieved, has been a focus of the field for many years. Recently, this laboratory has achieved an ~23% yield of electron transfer to the normally inactive M branch in a RC through targeted site-directed changes in the amino acids near BL, HL, and BM. The current work seeks to build on this progress as well as on parallel and equally important advances in understanding the factors underpinning the high yield of charge separation along the photoactive branch. Specifically, further site-directed mutations will be made in the RC protein and comprehensive studies of their effects on all the primary events carried out in order to achieve the following. (1) Elucidation of the participation of each cofactor in the primary events, the mechanisms of their involvement, and how the free-energy relationships of the associated charge-separated states impact the directionality of electron transfer and the rates and yields of charge separation versus charge recombination. (2) Understanding the manner in which polar and ionizable amino acid residues modulate the electronic and vibrational properties of the nearby cofactors and, thus, the photochemistry. (3) Deducing the contributions to directionality of the relative free energies and electronic couplings on the two branches (e.g. involving P* and P+BL- versus P+BM-). (4) Obtaining a greater yield of electron transfer both to the M branch (forming P+HM-) and along this branch (forming P+QB-) than has been achieved to date. This will open up many new and exciting avenues for work on P+QB- formed via the M-side versus the L-side and how the events on the two sides are manipulated by analogous mutations. Life on earth ultimately is dependent on photosynthesis, the process by which plants and certain bacteria convert the energy of sunlight into chemical potential energy. This conversion occurs via a series of fast electron transfer reactions that separate charge in a specialized membrane-bound pigment-protein complex called the reaction center (RC). Remarkably, charge separation in the RC takes place unidirectionally via one of two possible pathways and with a quantum yield of ~100%. This research project focuses on RCs from purple photosynthetic bacteria and seeks to come to a comprehensive molecular level understanding of this primary charge separation process. To this end, transient absorption spectroscopic measurements spanning the femtosecond (10-15 sec) to seconds time scales as well as other spectroscopic techniques will be used to directly probe the various stages of charge separation and the rates of formation and decay of the intermediates. These studies will provide further detailed insights into the molecular-level mechanism of one of the most important biological processes, aid in the development of biomimetics for solar energy conversion, and positively impact broader topics in biophysics including biological electron transfer and the role of a protein in modulating the functional properties of its cofactors.