The involvement of Arg265 of mouse ribonucleotide reductase R2 protein in proton transfer and catalysis.
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.
Narváez
Ana J
AJ
Department of Biochemistry and Biophysics, Stockholm University, SE-10691 Stockholm, Sweden.
Voevodskaya
Nina
N
Thelander
Lars
L
Gräslund
Astrid
A
D000818:Animals; D001120:Arginine / Q000737:chemistry*; D004579:Electron Transport / Q000502:physiology*; D007501:Iron / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011522:Protons; D012264:Ribonucleotide Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020349:Surface Plasmon Resonance
D021122:Protein Subunits; D011522:Protons; D001120:Arginine; D007501:Iron; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D010100:Oxygen