CYSTEINYL AND SUBSTRATE RADICAL FORMATION IN ACTIVE-SITE MUTANT E441QOF ESCHERICHIA-COLI CLASS-I RIBONUCLEOTIDE REDUCTASE

AU classes of ribonucleotide reductase are proposed to have a common r eaction mechanism involving a transient cysteine thiyl radical that in itiates catalysis by abstracting the 3'-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escher ichia coli we recently trapped two kinetically coupled transient radic als in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, R2, Katterle, B. , Potsch, S., Sahlin, M., and Sjoberg, B.-M. (1997) J. Biol. Chem. 272 , 31533-31541). Using isotopically labeled R1 protein or substrate, we now demonstrate that the early radical intermediate is a cysteinyl ra dical, possibly in weak magnetic interaction with the diiron site of p rotein R2, and that the second radical intermediate is a carbon-center ed substrate radical with hyperfine coupling to two almost identical p rotons. This is the first report of a cysteinyl free radical in ribonu cleotide reductase that is a kinetically coupled precursor of an ident ified substrate radical. We suggest that the cysteinyl radical is loca lized to the active site residue, Cys(439) which is conserved in all c lasses of ribonucleotide reductase, and which, in the three-dimensiona l structure of protein R1, is positioned to abstract the 3'-hydrogen a tom of the substrate. We also suggest that the substrate radical is lo calized to the 3'-position of the ribose moiety, the first substrate r adical intermediate in the postulated reaction mechanism.