Free radicals play key roles in a number of biological redox and metabolic reactions. In the research proposed here we will focus on two classes of these paramagnetic intermediates: (a) quinone free radicals which occur in several of the steps in mitochondrial and photosynthetic electron transfer and (b) tyrosine free radicals, which are prevalent as protein oxidation products, are also essential to catalysis in nucleic acid formation by the enzyme, ribonucleotide reductase (RDPR). In their protein binding sites, these radicals are immobilized and EPR lineshapes are badly distorted by anisotropic interactions which are not averaged. Our preliminary data indicate that ENDOR spectroscopy of these powder samples can be used to extract both isotropic and anisotropic interaction parameters for several kinds of protons which are present as substituents on the quinone ring. Moreover, ENDOR has the capability to explore the protein binding site at short range (less than 6 Angstrom) and to identify binding site/radical interactions. We plan to extend this approach by studying a series of quinone and tyrosine model compounds which contain a variety of biologically relevant ring substituents. Techniques for spectral assignment which complement the orientation selection and deuterium exchange we now use will be developed. These include Q-band EPR, ENDOR triple resonance, two-dimensional sample orientation and computational approaches. The in situ systems to be studied include the Z+/D+ radical involved in water oxidation, the tyrosine radical in RDPR and a variety of quinone radical intermediates in respiratory and photosynthetic systems. The techniques developed for the model compound studies will be used in this work; we will also substitute specific, isotopically labeled amino acids into some of the proteins under study, notably RDPR and the water splitting assembly, in order to facilitate spectral assignment. The underlying assumption in this work is that the control of free radical function in these systems is achieved through protein/radical interaction; the long term goal of this work is to characterize these contacts.
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