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

• Published in: The Journal of biological chemistry 1999-08, Vol.274 (34), p.23746
• Main Authors: Rova, U, Adrait, A, Pötsch, S, Gräslund, A, Thelander, L
• Format: Article
• Language: English
• Subjects: Animals; Catalysis; Free Radicals; Iron; Mice; Mutagenesis, Site-Directed; Ribonucleotide Reductases - chemistry; Structure-Activity Relationship; Tyrosine
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.34.23746

Ribonucleotide reductase catalyzes all de novo synthesis of deoxyribonucleotides. The mammalian enzyme consists of two non-identical subunits, the R1 and R2 proteins, each inactive alone. The R1 subunit contains the active site, whereas the R2 protein harbors a binuclear iron center and a tyrosyl free radical essential for catalysis. It has been proposed that the radical properties of the R2 subunit are transferred approximately 35 A to the active site of the R1 protein, through a coupled electron/proton transfer along a conserved hydrogen-bonded chain, i.e. a radical transfer pathway (RTP). To gain a better insight into the properties and requirements of the proposed 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 same chain in Y370W slows down the RTP, resulting in a 58 times lower specific activity compared with the native R2 protein and a loss of the free radical during catalysis.