Pulsed EPR Studies of Biological Manganese Clusters

简介（申请人提供）：过渡金属锰在生物学和医学中发挥着多种重要作用。例如，许多酶在其催化中心使用Mn（ll）。除了Mn（ll）外，更高的氧化态Mn（ll）和Mn（IV）也用于关键的氧中心氧化还原化学。特别令人感兴趣的是光系统II的出氧络合物（OEC）的四核Mn簇实现的独特的水氧化化学。该簇将近端酪氨酸自由基(Yz（）的高氧化电位与两个结合水的氧化结合在一起，在5-中间循环的最后一步释放分子氧。因此，这个系统是金属化学的一个重要例子，而且每种状态都可以用激光高产量地产生，这使得这个光合系统成为探索这种有趣化学的理想选择。我们将使用多频率（9、31、35和130 GHz）先进的电子顺磁共振（EPR）方法，包括ENDOR、ESEEM和HYSCORE来研究氧演化循环的中间体。这些实验将针对Mn簇的结构、氨基酸配位、Ca2+和Cl-辅因子的位置和功能以及底物水的结合。我们将遵循新的x射线晶体结构的线索，以针对当前高兴趣的特定问题。我们将使用高场/频率（130 GHz） EPR/ENDOR仪器对光系统II单晶中的Mn团簇进行高分辨率光谱分析。使用新组装的快速冷冻淬火系统，我们将在激光闪光序列后的毫秒时间尺度上冷冻样品，最终目标是表征OEC循环的最终短暂的s4状态。这项研究有望揭示重要的新细节，揭示生物金属簇如何以远高于我们现有技术的效率从水中产生分子氧。相关性：这项拨款提案的重点是了解这一重要的生命过程，它产生我们呼吸所需的大气中的氧气，并从生物角度激活植物所需的水中的电子和质子，将大气中的二氧化碳转化为我们的主要食物来源。

DESCRIPTION (provided by applicant): The transition metal manganese plays a variety of important roles in biology and medicine. For example, a large number of enzymes use Mn(ll) in their catalytic centers. Along with Mn(ll), the higher oxidation states, Mn(lll) and Mn(IV), are also used in crucial oxygen-centered redox chemistry. Of particular interest is the unique water oxidation chemistry enabled by the tetranuclear Mn cluster of the oxygen evolving complex (OEC) of photosystem II. This cluster couples the high oxidation potential of a proximal tyrosine radical (Yz() to the oxidation of two bound waters, releasing molecular oxygen at the final step of a 5-intermediate cycle. Thus this system is an important example of metalloradical chemistry, and the fact that each state can be generated in high yield with laser flashes makes this photosynthetic system ideal for exploring this intriguing chemistry. We will examine the intermediates of the oxygen evolving cycle with multifrequency (9, 31, 35, and 130 GHz) advanced electron paramagnetic resonance (EPR) methods, including ENDOR, ESEEM, and HYSCORE. These experiments will target the structure of the Mn cluster, its amino acid coordination, the location and function of the Ca2+ and Cl- cofactors, and the binding of substrate waters. We will follow leads from new x-ray crystal structures to target specific issues of high current interest. We will use our high field/frequency (130 GHz) EPR/ENDOR instrument to perform high resolution spectroscopy of the Mn cluster in single crystals of photosystem II. Using a newly assembled rapid freeze quench system, we will cryotrap samples on the millisecond timescale after laser flash sequences, with the ultimate goal of characterizing the final short-lived S4-state of the OEC cycle. This research promises to reveal important new details concerning how a biological metal cluster can produce molecular oxygen from water with an efficiency far greater than we can achieve with our current technologies. Relevance: This grant proposal focuses on understanding this vital life process, which produces the oxygen of our atmosphere that we require for respiration, and biologically activates the electrons and protons from water needed by plants to convert atmospheric carbon dioxide into our primary food sources.