NITRITE REDUCTION AN ESEEM STUDY

Ribonucleotide triphosphate reductase (RTPR) from L. leichmannii catalyzes the adenosylcobalamin-dependent reduction of nucleotides to deoxynucleotides. When the enzyme is rapidly mixed with adenosylcobalamin, an allosteric effector and a reducing system, a paramagnetic species is generated in a kinetically competent fashion. Although originally observed over 20 years ago, the identity of the radical species was previously unknown. Isotopic labeling of cystein in conjunction with simulations of this system have recently shown that the signal is consistent with a thiyl radical interacting with cob(II)alamin. However, much remains to be learned concerning the structure of this thiyl radical-cob(II)alamin pair. High frequency EPR and ENDOR spectroscopies will be used to give insight into the molecular and electronic structure of the radical pair. Refinement of the distance between unpaired electrons and the relative orientation of the thiyl radical and cob(II)alamin pair will give additional structural additional structural detail to the enzyme active site. A key question concerns the ate of the deoxyadenosyl moiety, since the 5'deosyadenosyl radical proposed to initially form upon Co-C bond homolysis has not been observed. Pulsed EPR/ENDOR in conjunction with 13C and 2H labeling at various positions on the 5'deoxyadenosine will determine its placement in the active site. In addition, RTPR is readily inhibited upon incubation with several different nucleotide analogs via unknown radical based mechanisms. Pulsed EPR, ENDOR and high frequency EPR spectroscopy will be performed in order to identify these radical intermediates and to determine the mechanisms of RTPR inhibition.