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
