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Abstract

Ribonucleotide reductase (RNR) catalyzes the biosynthesis of
deoxyribonucleotides. The active enzyme contains a diiron
center and a tyrosyl free radical required for enzyme activity.
The radical is located at Y177 in the R2 protein of mouse RNR.
The radical is formed concomitantly with the mu-oxo-bridged
diferric center in a reconstitution reaction between ferrous
iron and molecular oxygen in the protein. EPR at 9.6 and 285
GHz was used to investigate the reconstitution reaction in the
double-mutant Y177F/1263C of mouse protein R2. The aim was to
produce a protein-linked radical derived from the Cys residue
in the mutant protein to investigate its formation and
characteristics. The mutation Y177F hinders normal radical
formation at Y177, and the I263C mutation places a Cys residue
at the same distance from the iron center as Y177 in the native
protein. In the reconstitution reaction, we observed small
amounts of a transient radical with a probable assignment to a
peroxy radical, followed by a stable sulfinyl radical, most
likely located on C263. The unusual radical stability may be
explained by the hydrophobic surroundings of C263, which
resemble the hydrophobic pocket surrounding Y177 in native
protein R2. The observation of a sulfinyl radical in RNR
strengthens the relationship between RNR and another free
radical enzyme, pyruvate formate-lyase, where a similar
relatively stable sulfinyl radical has been observed in a
mutant. Sulfinyl radicals may possibly be considered as
stabilized forms of very short-lived thiyl radicals, proposed
to be important intermediates in the radical chemistry of RNR.