Cation mediation of radical transfer between Trp48 and Tyr356 during O ₂ activation by protein R2 of Escherichia coli ribonucleotide reductase: Relevance to R1-R2 radical transfer in nucleotide reduction

A tryptophan 48 cation radical (W48^+⊙) forms concomitantly with the Fe₂(III/IV) cluster, X, during activation of oxygen for tyrosyl radical (Y122^⊙) production in the R2 subunit of class I ribonucleotide reductase (RNR) from Escherichia coli. W48^+⊙ is also likely to be an intermediate in the long-range radical transfer between R2 and its partner subunit, R1, during nucleotide reduction by the RNR holoenzyme. The kinetics of decay of W48^+⊙ and formation of tyrosyl radicals during O₂ activation (in the absence of R1) in wild-type (wt) R2 and in variants with either Y122, Y356 (the residue thought to propagate the radical from W48^+⊙ into R1 during turnover), or both replaced by phenylalanine (F) have revealed that the presence of divalent cations at concentrations similar to the [Mg²⁺] employed in the standard RNR assay (15 mM) mediates a rapid radical-transfer equilibrium between W48 and Y356, Cation-mediated propagation of the radical from W48 to Y356 gives rise to a fast phase of Y^⊙ production that is essentially coincident with W48^+⊙ formation and creates an efficient pathway for decay of W48^+⊙. Possible mechanisms of this cation mediation and its potential relevance to intersubunit radical transfer during nucleotide reduction are considered.