Two conserved tyrosine residues in protein R1 participate in an intermolecular electron transfer in ribonucleotide reductase.
The enzyme ribonucleotide reductase consists of two nonidentical proteins, R1 and R2, which are each inactive alone. R1 contains the active site and R2 contains a stable tyrosyl radical essential for catalysis. The reduction of ribonucleotides is radical-based, and a long range electron transfer chain between the active site in R1 and the radical in R2 has been suggested. To find evidence for such an electron transfer chain in Escherichia coli ribonucleotide reductase, we converted two conserved tyrosines in R1 into phenylalanines by site-directed mutagenesis. The mutant proteins were shown to be enzymatically inactive. In addition, the mechanism-based inhibitor 2'-azido-2'-deoxy-CDP was incapable of scavenging the R2 radical, and no azido-CDP-derived radical intermediate was formed. We also show that the loss of enzymatic activity was not due to impaired R1-R2 complex formation or substrate binding. Based on these results, we predict that the two tyrosines, Tyr-730 and Tyr-731, are part of a hydrogen-bonded network that constitutes an electron transfer pathway in ribonucleotide reductase. It is demonstrated that there is no electron delocalization over these tyrosines in the resting wild-type complex.
Ekberg
M
M
Department of Molecular Biology, University of Stockholm, S-10691 Stockholm, Sweden.
Sahlin
M
M
Eriksson
M
M
Sjöberg
B M
BM
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D006153:Guanosine Diphosphate / Q000378:metabolism; D006860:Hydrogen Bonding; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000737:chemistry*
D017931:DNA Primers; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D014443:Tyrosine; D012264:Ribonucleotide Reductases