Cysteinyl and substrate radical formation in active site mutant E441Q of Escherichia coli class I ribonucleotide reductase.

All classes of ribonucleotide reductase are proposed to have a common reaction mechanism involving a transient cysteine thiyl radical that initiates catalysis by abstracting the 3'-hydrogen atom of the substrate nucleotide. In the class Ia ribonucleotide reductase system of Escherichia coli, we recently trapped two kinetically coupled transient radicals in a reaction involving the engineered E441Q R1 protein, wild-type R2 protein, and substrate (Persson, A. L., Eriksson, M., Katterle, B., Pötsch, S., Sahlin, M., and Sjöberg, 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 radical, possibly in weak magnetic interaction with the diiron site of protein R2, and that the second radical intermediate is a carbon-centered substrate radical with hyperfine coupling to two almost identical protons. This is the first report of a cysteinyl free radical in ribonucleotide reductase that is a kinetically coupled precursor of an identified substrate radical. We suggest that the cysteinyl radical is localized to the active site residue, Cys439, which is conserved in all classes of ribonucleotide reductase, and which, in the three-dimensional structure of protein R1, is positioned to abstract the 3'-hydrogen atom of the substrate. We also suggest that the substrate radical is localized to the 3'-position of the ribose moiety, the first substrate radical intermediate in the postulated reaction mechanism.