The Involvement of Arg265 of Mouse Ribonucleotide Reductase R2 Protein in Proton Transfer and Catalysis

Ana J Narváez,Nina Voevodskaya,Lars Thelander,Astrid Gräslund

doi:10.1074/jbc.m604598200

Ana J Narváez, Nina Voevodskaya + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m604598200

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Abstract

Ribonucleotide reductase class I enzymes consist of two non-identical subunits, R1 and R2, the latter containing a diiron carboxylate center and a stable tyrosyl radical (Tyr*), both essential for catalysis. Catalysis is known to involve highly conserved amino acid residues covering a range of approximately 35 A and a concerted mechanism involving long range electron transfer, probably coupled to proton transfer. A number of residues involved in electron transfer in both the R1 and R2 proteins have been identified, but no direct model has been presented regarding the proton transfer side of the process. Arg265 is conserved in all known sequences of class Ia R2. In this study we have used site-directed mutagenesis to gain insight into the role of this residue, which lies close to the catalytically essential Asp266 and Trp103. Mutants to Arg265 included replacement by Ala, Glu, Gln, and Tyr. All mutants of Arg265 were found to have no or low catalytic activity with the exception of Arg265 to Glu, which shows approximately 40% of the activity of native R2. We also found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2. Our results, supported by molecular modeling, strongly suggest that Arg265 is involved in the proton-coupled electron transfer pathway and may act as a proton mediator during catalysis.

Highlights

Ribonucleotide reductase (RNR)2 of the class I family catalyzes the reduction of nucleoside diphosphates to deoxynucleoside diphosphates and is essential for de novo synthesis of deoxyribonucleotides required for DNA replication and repair [1,2,3,4]
Using site-directed mutagenesis, a pathway at the mammalian RNR has been identified as the route for the electron transfer that leads from the cysteinyl radical in R1 through the residues Cys429-Tyr738-Tyr737 across the R1/R2 interface and in R2 through the residues Tyr370Trp103-Asp266 toward the iron site (16 –19)
proton-coupled electron transfer (PCET) reactions have been extensively studied in various systems including respiration and photosynthesis

Summary

Introduction

Ribonucleotide reductase (RNR)2 of the class I family catalyzes the reduction of nucleoside diphosphates to deoxynucleoside diphosphates and is essential for de novo synthesis of deoxyribonucleotides (dNTPs) required for DNA replication and repair [1,2,3,4]. We found that the Arg mutants were capable of stable tyrosyl radical generation, with similar kinetics of radical formation and R1 binding as native R2.

Results

Conclusion