2 CONSERVED TYROSINE RESIDUES IN PROTEIN R1 PARTICIPATE IN AN INTERMOLECULAR ELECTRON-TRANSFER IN RIBONUCLEOTIDE REDUCTASE

The enzyme ribonucleotide reductase consists of two nonidentical prote ins, R1 and R2, which are each inactive alone, R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis, The reduction of ribonucleotides is radical based, and a long range e lectron transfer chain between the active site in R1 and the radical i n R2 has been suggested, To find evidence for such an electron transfe r chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by site-directed mutage nesis. The mutant proteins were shown to be enzymatically inactive, In addition, the mechanism-based inhibitor 2'-azido-2'-deoxy-CDP was inc apable of scavenging the R2 radical, and no azido-CDP-derived radical intermediate was formed, We also shaw that the loss of enzymatic activ ity was not due to impaired R1-R2 complex formation or substrate bindi ng, Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogenbonded network that constitutes an electron transfer pathway in ribonucleotide reductase, It is demonstr ated that there is no electron delocalization over these tyrosines in the resting wild-type complex.