Kinetic evidence that a radical transfer pathway in protein R2 of mouse ribonucleotide reductase is involved in generation of the tyrosyl free radical

Class I ribonucleotide reductases consist of two subunits, R1 and R2, The active site is located in B1; active R2 contains a diferric center and a tyrosyl free radical (Tyr(.)), both essential for enzymatic activity. The proposed mechanism for the enzymatic reaction includes the transport of a reducing equivalent, i.e. electron or hydrogen radical, across a 35-Angstrom distance between Tyr(.) in R2 and the active site in R1, which are connected by a hydrogen-bonded chain of conserved, catalytically essential amino acid residues. Asp(266) and Trp(103) in mouse R2 are part of this radical transfer pathway. The differic/Tyr(.) site in R2 is reconstituted spontaneously by mixing iron-free apoR2 with Fe(II) and O-2. The reconstitution reaction requires the delivery of an external reducing equivalent to form the diferric/Tyr(.) site. Reconstitution kinetics were investigated in mouse ape-wild type R2 and the three mutants D266A, W103Y, and W103F by rapid freeze-quench electron paramagnetic resonance with greater than or equal to 4 Fe(II)/R2 at various reaction temperatures. The kinetics of Tyr(.) formation in D266A and W103Y is on average 20 times slower than in wild type R2, More strikingly, Tyr(.) formation is completely suppressed in W103F, No change in the reconstitution kinetics was found starting from Fe(II)-preloaded proteins, which shows that the mutations do not affect the rate of iron binding. Our results are consistent with a reaction mechanism using Asp266 and Trp(103) for delivery of the external reducing equivalent. Further, the results with W103F suggest that an intact hydrogen-bonded chain is crucial for the reaction, indicating that the external reducing equivalent is a H-.. Finally, the formation of Tyr(.) is not the slowest step of the reaction as it is in Escherichia coli R2, consistent with a stronger interaction between Tyr(.) and the iron center in mouse R2, A new electron paramagnetic resonance visible intermediate named mouse X, strikingly similar to species X found in E. coli R2, was detected only in small amounts under certain conditions. We propose that it may be an intermediate in a side reaction leading to a diferric center without forming the neighboring Tyr ..