EVIDENCE BY SITE-DIRECTED MUTAGENESIS SUPPORTS LONG-RANGE ELECTRON-TRANSFER IN MOUSE RIBONUCLEOTIDE REDUCTASE

Mammalian ribonucleotide reductase consists of two nonidentical subuni ts, proteins R1 and R2, each inactive alone. The R1 protein binds the ribonucleotide substrates while the R2 protein contains a binuclear ir on center and a tyrosyl free radical, essential for activity. The crys tal structures of the corresponding Escherichia coli proteins suggest that the distance from the active site in R1 to the tyrosyl radical bu ried in R2 is about 35 Angstrom. Therefore, an electron pathway was su ggested between the active site and the tyrosyl radical. Such a pathwa y could include a conserved tryptophan on the suggested R1 interaction surface of R2 and a conserved aspartic acid hydrogen bonded both to t he tryptophan and to a histidine iron ligand. To find experimental sup port for such an electron pathway, we have replaced the conserved tryp tophan in mouse R2 with phenylalanine or tyrosine and the aspartic aci d with alanine. All the mutated R2 proteins were shown to bind metal w ith the same affinity as native R2 and to form the binuclear iron cent er. In addition, the W103Y and D266A proteins formed a normal tyrosyl free radical while only low amounts of radical were observed in the W1 03F protein. Neither the kinetic rate constants nor the equilibrium di ssociation constant of the R1/R2 complex was affected by the mutations as shown by BIAcore biosensor technique. However, all mutant R2 prote ins were completely inactive in the enzymatic assay, supporting the hy pothesis that the tryptophan and aspartic acid residues are important links in an amino acid residue specific long-range electron transfer.