FORMATION OF A FREE-RADICAL OF THE SULFENYLIMINE TYPE IN THE MOUSE RIBONUCLEOTIDE REDUCTASE REACTION WITH 2'-AZIDO-2'-DEOXYCYTIDINE 5'-DIPHOSPHATE

Mouse and Escherichia coli ribonucleotide reductases (RR) both belong to the same class of RR, where the enzyme consists of two non-identica l subunits, proteins R1 and R2. A transient free radical was observed by EPR spectroscopy in the mouse RR reaction with the suicidal inhibit or 2'-azido-2'-deoxycytidine 5'-diphosphate. The detailed hyperfine st ructure of the EPR spectrum of the transient radical is somewhat diffe rent for the mouse and previously studied E. coli enzymes. When the po sitive allosteric effector ATP was replaced by the negative effector d ATP, no transient radical was observed, showing that 'normal' binding of the inhibitor to the substrate binding site is required. Using the mouse protein R2 mutants W103Y and D266A, where the mutations have bee n shown to specifically block long range electron transfer between the active site of the R1 protein to the iron/radical site in protein R2, no evidence of transient radical was found. Taken together, the data suggest that the radical is located at the active site in protein R1, and is probably of the sulfenylimine type.