Evidence by mutagenesis that Tyr(370) of the mouse ribonucleotide reductase R2 protein is the connecting link in the intersubunit radical transfer pathway

Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleo tides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active si te, whereas the R2 protein harbors a binuclear iron center and a tyrosyl fr ee radical essential for catalysis. It has been proposed that the radical p roperties of the R2 subunit are transferred similar to 35 Angstrom to the a ctive site of the R1 protein, through a coupled electron/proton transfer al ong a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP ). To gain a better insight into the properties and requirements of the pro posed RTP, we have used site directed mutagenesis to replace the conserved tyrosine 370 in the mouse R2 protein with tryptophan or phenylalanine. This residue is located close to the flexible C terminus, known to be essential for binding to the R1 protein. Our results strongly indicate that Tyr(370) links the RTP between the R1 and R2 proteins. Interruption of the hydrogen -bonded chain in Y370F inactivates the enzyme complex. Alteration of the sa me chain in Y370W slows down the RTP, resulting in a 58 times lower specifi c activity compared with the native R2 protein and a loss of the free radic al during catalysis.