HIGH FREQUENCY CRYOGENIC EPR STUDY OF METHYLMALONYL COA MUTASE ENZYME

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X-band EPR studies on methylmalonyl-CoA mutase in Prof. Banerjee's laboratory show rather messy-looking spectra that seems to represent contributions from two separate species (JBC, 1995, 270:9295; JACS, 1995, 117:7033). This system requires additional information for a definitive interpretation. The Banerjee group has between 60 and 80 uM spin associated with the catalytic intermediate, which has a crossover g value at 2.11. By selective isotope substitution studies, the signals are deduced to show a biradical intermediate which has an exchange coupled Co(II)-organic radical pair. This is a steady-state sample, and they have never been able to get more than 0.4 spin/mol of enzyme. High-field EPR is being undertaken with the hope that we can separate the signals and eventually simulate the spectra. Sample handling is challenging, as the enzyme must be handled anaerobically prior to freezing, and the capillary sample tubes are difficult to load, ship, preserve in frozen state, and load ed into the spectrometer without thawing or contamination. Nevertheless, progress is being made, and preliminary Q-band and W-band spectra have been obtained at the IERC. This project will continue in 1998.