The iron-oxygen reconstitution reaction in protein R2-tyr-177 mutants of mouse ribonucleotide reductase - EPR and electron nuclear double resonance studies on a new transient tryptophan radical

The ferrous iron/oxygen reconstitution reaction in protein R2 of mouse and Escherichia coli ribonucleotide reductase (RNR) leads to the formation of a stable protein-linked tyrosyl radical and a mu-oxo-bridged diferric iron c enter, both necessary for enzyme activity. We have studied the reconstituti on reaction in three protein R2 mutants Y177W, Y177F, and Y177C of mouse RN R to investigate if other residues at the site of the radical forming Tyr-1 77 can harbor free radicals. In Y177W we observed for the first time the fo rmation of a tryptophan radical in protein R2 of mouse RNR with a Lifetime of several minutes at room temperature. We assign it to an oxidized neutral tryptophan radical on Trp-177, based on selective deuteration and EPR and electron nuclear double resonance spectroscopy in H2O and D2O solution. The reconstitution reaction at 22 degrees C in both Y177F and Y177C leads to t he formation of a so-called intermediate X which has previously been assign ed to an oxo (hydroxo)-bridged Fe(III)/Fe(IV) cluster. Surprisingly, in bot h mutants that do not have successor radicals as Trp' in Y177W, this cluste r exists on a much longer time scale (several seconds) at room temperature than has been reported for X in E. coli Y122F or native mouse protein R2. A ll three mouse R2 mutants were enzymatically inactive, indicating that only a tyrosyl radical at position 177 has the capability to take part in the r eduction of substrates.