Tryptophan radicals formed by iron/oxygen reaction with Escherichia coli ribonucleotide reductase protein R2 mutant Y122F.

Abstract The active state of the small subunit, protein R2, of ribonucleotide reductase is formed by the reaction of apoprotein with Fe2+ and O2, whereby the diferric site and a stable phenoxy free radical on a tyrosyl residue (Tyr122) is formed. The corresponding reaction was studied in the mutant Y122F R2. It leads to a normal iron site, but the reduction equivalent from Tyr122 now has to be supplied from elsewhere. EPR spectroscopy shows formation of several paramagnetic species on different time scales. Using apoprotein with deuterium-labeled tryptophan residues, at least two species could be assigned to tryptophan free radicals. This is the first EPR observation of relatively stable protein-linked tryptophan radicals at room temperature and at 77 K. These tryptophan radicals may be involved as redox intermediates in long range electron transfer within the protein structure.