HANDLING OF THIYL METALLOENZYME CENTERS FOR 95 & 140 GHZ EPR STUDIES

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The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii requires adenosylcobalamin (AdoCbl) as a cofactor to catalyze the conversion of nucleotides to deoxynucleotides. RTPR has previously been shown to catalyze the homolytic cleavage of the carbon-cobalt bond of AdoCbl, and the resulting paramagnetic species has been characterized by rapid freeze-quench X-band and Q-band spectroscopy (Orme-Johnson, W. Fl.; Beinert, H.; Blakeley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, G.J.; Stubbe, J. Science 1996, 271, 477-481). Simulations indicate a two-spin-site system with both cobalt ion and thiyl radical (Gerfen, G.J.; Licht, S.; Willems, J.P.; Hoffman, B.R.; Stubbe, J. JACS 1996, 18:2338-2343), but higher EPR frequencies are needed for definitive conclusions on this and other B12-dependent enzyme systems. Sample preparation, handling, and loading is challenging for the small sample tubes (less than 1 mm ID) of the 95 GHz and 140 GHz EPR spectromete rs at IERC and MIT, respectively. This sample handling problem is being addressed.