Acquisition of a Pulsed FT-EPR Spectrometer

建议购买商业制造的电子顺磁共振（EPR）仪器，这将推动亚利桑那州立大学光合作用中心的EPR实验室在几个领域达到最先进的水平。大部分预算用于为该中心现有的连续波（CW） EPR光谱仪（Bruker ESP 300E）购买脉冲傅里叶变换(FT)-EPR配件Bruker ESP 380E。380E提供了更高的灵敏度、时间分辨率、可靠性、易用性和（最重要的）通用性，超过了目前中心研究人员使用的自制脉冲光谱仪。例如，380E可以进行二维电子自旋回波包络调制（ESEEM）实验，自动执行复杂的相位循环例程，并且无需修改微波或脉冲定时硬件即可实现长或新颖的脉冲序列。该中心目前的脉冲仪器没有这些功能。此外，将购买300E和380E的附件，首次为中心的研究人员提供以下光谱工具：脉冲电子-核双共振（脉冲ENDOR）；35 GHz EPR测量范围从3.8 K到室温；和35ghz ENDOR。该中心的布鲁克300E连续波光谱仪的拟议升级还将包括（由PI）建造Lubitz描述的高q石英9 GHz ENDOR腔。该仪器的用途将主要在光合作用和植物科学领域，但也将包括在生命科学中应用的蛋白质结构和酶机制的基础研究。例如，脉冲和连续波ENDOR以及快速时间分辨EPR测量将有助于表征酪氨酸D或Z自由基性质受到影响的光系统II突变体。在使用氧化钒（VO2+）和锰（Mn2+）作为顺磁探针的叶绿体fl - atp酶研究中，新仪器的可用性将允许精确测定局部配体结构。此外，已经确定晶体结构的金属配体模型系统的广泛光谱表征将有助于建立常见的基于蛋白质的金属配体的“签名”超精细耦合。确定atp酶催化周期中的结构变化将导致其高度复杂的催化机制的完整映射。使用新仪器检查的其他系统有：光系统I；光合绿硫细菌中的Fenna-Matthews-Olson蛋白；赖氨酸2,3-氨基转化酶和乙醇胺-氨裂解酶（两者的反应都是通过自由基中间体进行的）；s -腺苷甲硫氨酸合成酶，在许多生物体中产生主要的细胞内甲基化剂。最后，FT-EPR将对人工光合系统中的短寿命电荷分离态进行测量，以探索结构对电子转移过程影响的复杂问题。在所有情况下，所提出的仪器将在某种程度上从无序样品中获得原子尺度的结构信息，这是迄今为止不可能的。拟议的设备采购/建设资金将大大加强研究生的研究训练以及本科生的暑期研究经历。脉冲仪器的易用性的提高将使得在正式课程的背景下或作为个人实验室培训的一部分，向学生介绍脉冲技术的复杂性更加实际。该项目还将使该中心的EPR设施与全国领先机构的研究人员保持高调的外部合作，并吸引其他此类合作。)A - I

The purchase of commercially-built electron paramagnetic resonance (EPR) instrumentation is proposed which will advance the ASU Photosynthesis Center's EPR laboratory to the state of the art in several areas. The majority of the budget is allocated to the purchase of a pulsed Fourier Transform (FT)-EPR accessory, the Bruker ESP 380E, for the Center's existing continuous-wave (CW) EPR spectrometer, a Bruker ESP 300E. The 380E offers improved sensitivity, time resolution, reliability, ease of use, and (most importantly) versatility over the home-built pulsed spectrometer currently used by the Center's investigators. For example, 380E can perform two-dimensional electron spin echo envelope modulation (ESEEM) experiments, execute complex phase-cycling routines automatically, and implement long or novel pulse sequences without modification of microwave or pulse timing hardware. The Center's present pulsed instrument has none of these capabilities. In addition, attachments for the 300E and 380E will be purchased that will provide the Center's investigators, for the first time, with the following spectroscopic tools: pulsed electron-nuclear double resonance (pulsed ENDOR); 35 GHz EPR measurements from 3.8 K to room temperature; and 35 GHz ENDOR. The proposed upgrade of the Center's Bruker 300E CW spectrometer will also include construction (by the PI) of a high-Q quartz 9 GHz ENDOR cavity of the type described by Lubitz. The uses of this instrumentation will lie mainly in the area of photosynthesis and plant science, but will also include basic studies of protein structure and enzyme mechanisms which have applications across the life sciences. For example, pulsed and CW ENDOR and fast time-resolved EPR measurements will aid in characterization of Photosystem II mutants in which the properties of the tyrosine D or Z radicals have been affected. In studies of chloroplast Fl-ATPase using oxovanadium (VO2+) and Mn2+ as paramagnetic probes, the availability of the new in struments will permit precise determination of local ligand structure. In addition, extensive spectroscopic characterization of metal-ligand model systems whose crystal structures have been determined will help to establish the 'signature' hyperfine couplings from commonly-occurring protein-based metal ligands. Determination of structural changes during the ATPase's catalytic cycle will lead to full mapping of its highly complex catalytic mechanism. Other systems to be examined using the new instrumentation are: Photosystem I; the Fenna-Matthews-Olson protein in photosynthetic green sulfur bacteria; lysine 2,3-aminomutase and ethanolamine-ammonia lyase (both of whose reactions proceed through free radical intermediates); and the enzyme S-adenosylmethionine synthetase, which produces the primary intracellular methylating agent in a wide variety of organisms. Finally, FT-EPR measurements on short-lived charge-separated states in artificial photosynthetic systems will be undertaken, as a probe of the complex problem of the influence of structure upon electron transfer processes. In all cases, the proposed instrumentation will yield, to a degree not heretofore possible, atomic-scale structural information from disordered samples. Funding of the proposed equipment purchases/construction will enhance significantly the research training for graduate students as well as summer research experiences for undergraduates. The improvement in ease of use of the pulsed instrument will make it far more practical to introduce students to the intricacies of pulsed techniques, within the context of a formal course or as a part of individual laboratory training. This-project will also allow the Center's EPR facility to maintain its high-profile external collaborations with investigators from leading institutions nationwide, and to attract other such collaborations. ) A - I