TY - JOUR
T1 - Electron paramagnetic resonance investigations of a kinetically competent intermediate formed in ribonucleotide reduction
T2 - Evidence for a thiyl radical-cob(II)alamin interaction
AU - Gerfen, G. J.
AU - Licht, S.
AU - Willems, J. P.
AU - Hoffman, B. M.
AU - Stubbe, J.
PY - 1996/1/1
Y1 - 1996/1/1
N2 - 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 EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.
AB - 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 EPR spectroscopy (Orme-Johnson, W.H.; Beinert, H.; Blakley, R.L., J. Biol. Chem. 1974, 249, 2338-2343. Licht, S.; Gerfen, O.J.; Stubbe, J. Science 1996, 271, 477-481). This study presents simulations of X- and Q-band EPR spectra of this intermediate. Modeling this species as a thiyl radical coupled to cob(II)alamin by electron-electron exchange and dipolar interactions yields reasonable fits to spectra obtained at both microwave frequencies, whereas simulations that employ a single-spin model do not. This modeling provides support for the intermediacy of a thiyl radical in this system. The techniques employed here may prove generally useful in simulation of similar spectra observed in other B12-dependent enzyme systems.
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U2 - 10.1021/ja960363s
DO - 10.1021/ja960363s
M3 - Article
AN - SCOPUS:0029841914
SN - 0002-7863
VL - 118
SP - 8192
EP - 8197
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 35
ER -