Protein Control of Cofactor Function

光合反应中心（RC）中的光能转换涉及初始电子转移，随后是稳定电荷分离的几个步骤。在类球红细菌的RC中，初级和次级电子受体醌QA和QB在化学上是相同的（均为泛醌-10），但表现出非常不同的性质。因此，它们为理解蛋白质如何决定结合辅因子的性质提供了理想的模型。本研究的重点是不同性质的QA和QB，这决定了醌之间的电子和质子转移反应的性质（动力学和能量学）的起源。具体项目将研究：（1）小弱酸如何重新激活（2）在残基M265处的QA位点突变体，通过X射线结构测定、对Zn取代的RC的ENDOR和脉冲EPR研究以及通过计算能量最小化来进一步表征;（3）QA-半醌IR光谱的起源-“阴离子带”-使用具有显著和特征性改变的QA- IR光谱的M265突变体;（4）醌取代基在使用合成类似物确定QA和QB功能中的作用;（5）用诱变、功能分析和FTIR研究了近端和远端QB位点在第一次电子转移中的作用;（7）第二次电子转移的温度依赖性中的热力学和熵的贡献。在所有的项目中，自由能水平将通过延迟荧光，动力学和平衡的结果通过吸收光谱，和结构的影响通过FTIR，X射线衍射和ENDOR光谱来确定。催化对生命是绝对必要的，几乎所有的代谢催化都是由蛋白质酶进行的。然而，真正了解催化机制的酶很少，如果有的话。这主要是因为潜在的原子/分子相互作用的可观测性有限。然而，促进酶作用于底物的蛋白质-配体相互作用与修饰辅因子性质的蛋白质-配体相互作用相同，例如辅基和辅酶，并且其中许多是光谱学上赋予的。本研究描述了对光合反应中心（RC）的研究，该中心具有8个光谱活性辅助因子，呈高度对称排列，但功能不对称性很强。因此，它提供了一个特殊的系统，用于识别蛋白质-辅因子相互作用负责化学相同的辅因子的不同功能。除了至少8种不同的电子转移反应外，RC还进行质子转移、底物（醌）结合和化学反应以及蛋白质（细胞色素）识别、对接和结合。这个项目的智力价值是有机会在微观层面上观察，理解和操纵生物机制独特性背后的相互作用。更广泛的影响来自于这些属性，使这个项目成为培训青年科学家的一个特殊项目，具有实质性的多学科组成部分。预计这些结果将有助于更深入地了解光合作用和呼吸作用-这些过程对全球和人类的生存和健康至关重要，并为课堂教学，指导和激励学生以及帮助和教唆高中教师奠定了坚实的基础。

Light energy conversion in the photosynthetic reaction center (RC) involves an initial electron transfer, followed by several more steps that stabilize the charge separation. The primary and secondary electron acceptor quinones, QA and QB, in the RC of Rhodobacter sphaeroides are chemically identical (both ubiquinone-10) but exhibit very distinct properties. Thus, they provide an ideal model for understanding how proteins determine the properties of bound cofactors. This research focuses on the origins of the distinct properties of QA and QB, which determine the nature (kinetics and energetics) of the electron and proton transfer reactions between the quinones. Specific projects will study: (1) how small weak acids reactivate ("rescue") a wide variety of mutants that are impaired in proton uptake and proton transfer to QB; (2) QA site mutants at residue M265, to be further characterized by X-ray structure determination, ENDOR and pulsed EPR studies on Zn-substituted RCs, and by computational energy minimization; (3) the origin of the QA- semiquinone IR spectrum - the "anion band" - using M265 mutants which have striking and characteristically altered QA- IR spectra; (4) the role of quinone substituents in determining QA and QB function, using synthetic analogues; (5) the role of the proximal and distal QB sites in the 1st electron transfer, using mutagenesis, functional assays and FTIR; (7) the enthalpic and entropic contributions to the temperature dependence of the 2nd ET. In all projects, free energy levels will be determined by delayed fluorescence, kinetic and equilibrium consequences by absorption spectroscopy, and structural implications by FTIR, X-ray diffraction and ENDOR spectroscopy.Catalysis is absolutely essential to life, and almost all metabolic catalysis is performed by proteinaceous enzymes. However, the catalytic mechanism is truly understood for very few enzymes, if any. This is largely because of the limited observability of the underlying atomic/molecular interactions. However, the protein-ligand interactions that facilitate enzyme action on a substrate are the same as those that modify cofactor properties, such as prosthetic groups and coenzymes, and many of these are spectroscopically well-endowed. This research describes studies on the photosynthetic reaction center (RC), which has 8 spectroscopically-active cofactors in a highly symmetrical arrangement, but with strong functional asymmetry. It therefore provides an exceptional system for identifying the protein-cofactor interactions responsible for the different functionality of chemically identical cofactors. In addition to at least 8 different electron transfer reactions, the RC also performs proton transfers, substrate (quinone) binding and chemistry, and protein (cytochrome) recognition, docking and binding. The intellectual merit of this project is the chance to observe, understand and manipulate, at a microscopic level, the interactions that underlie the uniqueness of biological mechanisms. The broader impact derives from these attributes, which make this project an exceptional one for training young scientists, with a substantially multidisciplinary component. The results are expected to contribute to a deeper understanding of photosynthesis and respiration - processes that are central to global and human survival and health, and which have formed a strong basis for teaching, mentoring and motivating students in the classroom, as well as aiding and abetting high school teachers.