Electron Paramagnetic Resonance of Bovine Superoxide Dismutase.

Abstract

This thesis describes the design and construction of a system to record and analyse electron paramagnetic resonance data from single crystals, and presents the results obtained by applying this system to the study of the Cu(II) ion in bovine superoxide dismutase. The electron paramagnetic resonance (EPR) machine described was designed specifically for work with protein single crystals at 9.5 GHz. To achieve high sensitivity, the design incorporates a rectangular TE(,101) microwave resonant cavity. "Two circle" rotational freedom is provided by a magnet with a rotating base and a sample goniometer which incorporates a 72-tooth antibacklash wormwheel. A unique combination of hardware and "software" allows crystal axis alignment information obtained by X-ray diffraction precession photography to be used in orienting crystals for the EPR experiments. Since the X-ray diffraction camera sample mount is the EPR sample mount, the orientation of the crystal with respect to the magnetic field of the EPR experiment is known with a very high degree of accuracy. By using this system, EPR spectra have been obtained from crystals of bovine superoxide dismutase (BSD). Spectra have also been obtained from crystals of BSD containing isotopically pure ('65)Cu, and from crystals which have been reacted with cyanide (BSD + CN). To analyse the spectra, computer programs have been written which make use of an algorithm based on a second-order perturbation theory treatment of the spin Hamiltonian of Cu(II). These programs make use of interactive computer graphics to compare simulated crystal EPR spectra with the experimental data. Programs have also been written to simulate EPR spectra obtained from frozen solutions of BSD and BSD + CN, using the same computational techniques. The results of this detailed study of the EPR properties of the Cu(II)ion in BSD are interpreted in terms of the known crystallographicstructure of the protein. Specifically, the orientation of the principal(, )axes of the Cu(II) magnetic tensors (g and A) are described with respectto the coordinates of the atoms which make up the metal binding site of the protein. A shift in the direction and magnitude of g(,z) is observed upon binding of CN to the protein; this indicates that the active site may undergo a conformational change upon CN binding.