Controlling the Pathway of Electron Transfer in Bacterial Reaction Centers

光合作用反应中心内的光引发的电子传递是光合作用生物进行光能转换的首要事件。紫色非硫细菌的光合作用反应中心的核心是一个准对称的异二聚体，为跨膜电子传递提供了两条潜在的途径。过去的测量表明，在用红光或近红外光激励时，两条路径中只有一条(A侧)在任何程度上都被使用。最近，伍德伯里博士发现，用蓝光激发反应中心的Soret带，可以在200秒的激光脉冲范围内沿着交替的或B侧路径产生光化学。形成的态的光谱特征以及它们在低温下的长(纳秒)寿命，是涉及B侧细菌叶绿素的阴离子的电荷分离态的特征。这为一系列全新的测量打开了大门，这些测量试图了解反应中心B侧电子转移的机制和功能。这项研究的中心是电荷分离态的自由能和用于激发的光子能量之间的相互作用。电荷分离态的自由能可以通过改变P/P+势的突变体在很大范围内改变。初步数据表明，B侧电子转移的能量学越有利，启动B侧转移所需的光子能量就越低。B侧电子转移的生理功能尚不清楚。一种可能性是，它可以迅速熄灭反应中心辅因子和周围色氨酸残基吸收蓝光或紫外光形成的激发态。计划进行几项涉及破坏B侧转移的突变体的实验，以确定在没有功能性B侧光化学的情况下，反应中心是否更容易受到紫外线和蓝光的破坏。最后，为了产生一系列具有光谱定义的功能特性(损失或A侧电子转移、增加B侧转移等)的新突变体，伍德伯里博士将开发一种定向进化方法，该方法利用完全光学的手段从大型反应中心突变体库中筛选和选择细胞。

Light-initiated electron transfer within the photosynthetic reaction center is the primary event in solar energy conversion by photosynthetic organisms. The core of the photosynthetic reaction center from purple nonsulfur bacteria is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near infrared light. Recently, Dr. Woodbury has found that excitation with blue light into the Soret band of the reaction center gives rise to photochemistry along the alternate or B-side pathway apparently within a 200 fs laser pulse. The spectral signature of the states formed, and their long (nanosecond) lifetime at low temperature, are characteristic of charge separated states involving the anion of the B-side bacteriopheophytin. This opens the door to a whole new array of measurements attempting to understand both the mechanism and the function of B-side electron transfer in the reaction center. This research project centers around the interplay between the free energy of the charge separated states, which can be varied over a large range using mutants that alter the P/P+ potential, and the energy of the photon used in excitation. Preliminary data suggests that the more favorable the energetics of B-side electron transfer, the lower the energy of the photon that is required to initiate B-side transfer. The physiological function of B-side electron transfer is not yet known. One possibility is that it could rapidly quench the excited states formed from blue or UV light absorption by reaction center cofactors and surrounding tryptophan residues. Several experiments involving mutants that disrupt B-side transfer are planned to see if reaction centers are more susceptible to UV and blue light damage without functional B-side photochemistry. Finally, in order to generate a series of new mutants with spectrally defined functional properties (loss or A-side electron transfer, increased B-side transfer, etc.), Dr. Woodbury will develop a directed evolution methodology that utilizes entirely optical means of both screening and selecting cells from a large library of reaction center mutants.