| Nearly all known life depends on photosynthesis for the primary capture of energy available in the biosphere. The photosynthetic reaction center (RC) is the core protein in the process of converting solar energy into chemical energy. The RC functions by absorbing photons directly, or accepting excitons from antennae pigment complexes, to excite the special pair, P. With a time constant of 3 ps, an electron is transferred from P to HL, the bacteriopheophytin on the active branch of chromophores. Charge separation continues as the electron is transferred to QA (tau = 200 ps), then to QB (tau = 100 micros). Following the sequential absorption of two photons, there is a 2-electron reduction of the mobile quinone (QB) to quinol (QBH2). The quinol leaves the RC, enters the quinone pool found in the hydrophobic portion of the lipid bilayer membrane, and eventually deposits the H+ ions on the opposite side of the membrane thereby generating a proton gradient which powers ATP synthase.;The role of the accessory bacteriochlorophyll, BL, has long been debated. It is located between the primary electron donator, P, and the first directly observable electron acceptor, HL, of the charge separation process. A long-standing debate developed about whether the initial charge separation to HL proceeds by the limiting cases of a 1-step or 2-step process. In the case of the 1-step process, the P+B L- state exists only as a virtual state to facilitate rapid electron transfer between P and HL over the 10 A minimum edge-to-edge distance between those chromophores. The 2-step process consists of electron transfer to BL to form P+BL - as an intermediate followed by sub-picosecond electron transfer to HL.;Herein it is shown that the charge separated state P+B L- can be generated in mutant RCs as a sufficiently long-lived state to allow the measurement of its spectrum by transient absorption spectroscopy. In mutants designated DLL-2 and DLL-3, in which the HL chromophore was knocked out thereby preventing electron transfer beyond BL, P+BL - was generated with a lifetime of 500 ps. The bacteriochlorophyll anion (BL-) in RC was found to have a distinct band at 1017 nm, consistent with bacteriochlorophyll anions generated in organic solvent. This work shows that the P+BL- state is accessible from P*, and provides further evidence for the two-step mechanism. It must be noted that the HL-less mutants utilized a set of 10 mutations to generate the phenotype. To make more direct inferences on wild-type RC, a minimal set of mutations will need to be determined.;In the course of study of the DLL mutants it was found that some mutants had P* populations that decayed as two distinct subpopulations. Based on simulations the orientation of the hydroxyl of Tyr at M208 was believed to possibility be the root cause of these subpopulations. Mutations were made to restrict to a single orientation Y(M208) the only Tyr not hydrogen bonded in the RC, by addition of a hydrogen bond. One of these engineered mutants, I(M204)E may have successfully introduced the desired hydrogen bond as suggested by the 13C NMR spectrum of [4'-13C]Tyr enriched I(M204)E RCs. Experimentation in this area is ongoing.;Serendipitously, in this exploratory search the mutant I(M204)Q was found to knockout the BL chromophore. The removal of BL has been verified by pigment extraction, cryogenic absorption spectroscopy, ultrafast transient absorption, and combining I(M204)Q with other phenotypically distinct mutations. The I(M204)Q mutant now serves as a minimally perturbative mutation to halt electron transfer on L-branch of chromophores and permits the study of electron transfer on the M-branch. |
