This proposal outlines research in three areas associated with high frequency dynamic nuclear polarization (DNP) and electron spin resonance (ESR) and electron nuclear double resonance (ENDOR). (1) High frequency CW and pulsed DNP methods will be developed and employed to enhance signal strengths in MAS NMR spectra of model systems and large proteins. The experiments will utilize a 140 GHz gyrotron oscillator, whose construction is now complete, and will be applicable to proteins containing in-situ paramagnetic centers, such as tyrosyl radicals, and to diamagnetic proteins with paramagnetic dispersed in the solvent. (2) 140 GHz pulsed ESR experiments will be used to study the frequency, concentration and temperature dependence of the electron spin lattice relaxation (T1e) in model systems for DNP experiments and in proteins. The data are useful for optimizing the rate and magnitude of polarization enhancements, in determining structures of proteins containing free radicals and paramagnetic centers, and are inherently interesting. (3)140 GHz CW ESR and ENDOR investigations are planned for (a) a series of model compounds necessary to be studied with pulsed and CW DNP experiments; (b) a large and expanding class of redox proteins containing tyrosyl radicals -- ribonucleotide reductase, cytochrome-c peroxidase, galactose oxidase, etc. -- where we plan to determine the utility of high field spectra for identifying radical species and the structure of radicals formed on reaction with inhibitors; (c) Mn2+ containing proteins, such as ras-p2l, where we will employ the spectral narrowing which occurs at high field to detect linewidth contributions from 17O-labeled ligands, and (d) photosynthetic reaction centers, where we will determine the molecular orientation of g-tensors from single crystal studies.
