Evidence by Mutagenesis that Tyr370 of the Mouse Ribonucleotide Reductase R2 Protein Is the Connecting Link in the Intersubunit Radical Transfer Pathway

Ulrika Rova,Annie Adrait,Stephan Pötsch,Astrid Gräslund,Lars Thelander

doi:10.1074/jbc.274.34.23746

Ulrika Rova, Annie Adrait + Show 3 more

Open Access

https://doi.org/10.1074/jbc.274.34.23746

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Abstract

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.

Highlights

All ribonucleotide reductases characterized up to now use free radical chemistry to catalyze the de novo synthesis of deoxyribonucleotides from ribonucleotides [1, 2]
Radical Transfer in Mouse Ribonucleotide Reductase from the N-terminal end does not affect the specific activity of the recombinant mouse R2 protein
The two commonly discussed models for electron transfer over distances of up to 20 Å argue whether or not the intervening protein structure is an important factor for the efficiency of the electron transfer [36, 37]

Summary

Introduction

All ribonucleotide reductases characterized up to now use free radical chemistry to catalyze the de novo synthesis of deoxyribonucleotides from ribonucleotides [1, 2]. The small subunit, the R2 protein, does not bind substrate, and its function during catalysis is to transfer radical properties to the R1 protein toward the activation of the substrate [4] For this purpose, the R2 protein harbors a tyrosyl free radical generated from a binuclear, non-heme iron center in a reaction that requires oxygen [5,6,7]. To explain the involvement of the R2 tyrosyl radical in catalysis, a long-range electron transfer pathway between the two subunits was suggested involving the following conserved residues (mouse numbering): (Fe1), His173,Asp266,Trp103 in the R2 protein and Tyr738,Tyr737, Cys429 in the R1 protein [10, 11]. Peptides and peptidomimetics corresponding to the R2 protein C terminus inhibit ribonucleotide reductase (24 –27) They may have a dual function by both interfering with R1-R2 complex formation and the hydrogen radical transfer between the subunits

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