Pulsed EPR Studies of Biological Manganese Clusters

我们将采用先进的EPR方法研究光系统II放氧复合物的四核锰簇的结构和功能。 多频CW和脉冲EPR和ENDOR实验将被用来表征氧释放循环的S态中间体（S 0，S1，S2，S3）的物理和电子结构。ENDOR和ESEEM实验使用PSII颗粒掺入2 H-和19 F-标记的酪氨酸将确定YZ酪氨酸和Mn簇之间的距离在氧释放PSII颗粒。 ESEEM和多频ENDOR实验将研究底物，底物类似物，和抑制剂结合的Mn簇在不同的S状态的细节，以及接近的基本辅因子Ca 2+和Cl-。 17，23，和33 kDa的外源性多肽在限制访问较大的底物类似物和抑制剂的Mn簇的作用将进行探讨。 其他ESEEM和多频率ENDOR实验将研究在各种S状态下簇的氨基酸连接。 并行模式和高频EPR，沿着与ESEEM和ENDOR，将用于检测和表征信号从锰簇组装中间体和簇还原产物。已知结构的合成Mn模型簇的研究将使我们能够将EPR/ENDOR衍生的性质与已知的结构元素相关联。 我们还将表征感兴趣的锰配合物的结构，作为“光谱模型”的PSII锰簇。 最后，我们将针对单核锰酶，锰超氧化物歧化酶和锰脂氧合酶，和双核锰酶，锰过氧化氢酶，与我们先进的EPR方法进行研究。

We will employ advanced EPR methods to study the structure and function of the tetranuclear manganese cluster of the Photosystem II oxygen evolving complex. Multifrequency CW and pulsed EPR and ENDOR experiments will be used to characterize the physical and electronic structures of the S state intermediates (S0, S1, S2, S3) of the oxygen evolving cycle. ENDOR and ESEEM experiments using PSII particles incorporating 2H- and 19F-labelled tyrosine will determine the distance between the YZ tyrosine and the Mn cluster in oxygen-evolving PSII particles. ESEEM and multifrequency ENDOR experiments will investigate details of substrate, substrate analog, and inhibitor binding to the Mn cluster at different S states, as well as the proximity of essential cofactors Ca2+ and Cl-. The role of the 17, 23, and 33kDa extrinsic polypeptides in limiting access of larger substrate analogs and inhibitors to the Mn cluster will be explored. Other ESEEM and multifrequency ENDOR experiments will investigate the amino acid ligation of the cluster at various S states. Parallel mode and high frequency EPR, along with ESEEM and ENDOR, will be used to detect and characterize signals from Mn cluster assembly intermediates and cluster reduction products. Studies of synthetic Mn model clusters of known structure will allow us to correlate EPR/ENDOR derived properties with known structural elements. We will also characterize the structures of interesting Mn complexes that serve as "spectral models" for the PSII Mn cluster. Finally, we will target the mononuclear Mn enzymes, Mn superoxide dismutase and Mn lipoxygenase, and the dinuclear Mn enzyme, Mn catalase, for study with our advanced EPR methods.