2,3-difluorotyrosine at position 356 of ribonucleotide reductase R2: a probe of long-range proton-coupled electron transfer.
Escherichia coli class I ribonucleotide reductase catalyzes the conversion of ribonucleotides to deoxyribonucleotides and consists of two subunits: R1 and R2. R1 possesses the active site, while R2 harbors the essential diferric-tyrosyl radical (Y*) cofactor. The Y* on R2 is proposed to generate a transient thiyl radical on R1, 35 A distant, through amino acid radical intermediates. To study the putative long-range proton-coupled electron transfer (PCET), R2 (375 residues) was prepared semisynthetically using intein technology. Y356, a putative intermediate in the pathway, was replaced with 2,3-difluorotyrosine (F2Y, pKa = 7.8). pH rate profiles (pH 6.5-9.0) of wild-type and F2Y-R2 were very similar. Thus, a proton can be lost from the putative PCET pathway without affecting nucleotide reduction. The current model involving H* transfer is thus unlikely.
Yee
Cyril S
CS
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Chang
Michelle C Y
MC
Ge
Jie
J
Nocera
Daniel G
DG
Stubbe
JoAnne
J
D000595:Amino Acid Sequence; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011522:Protons; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism*
D011522:Protons; C025200:3,5-difluorotyrosine; D014443:Tyrosine; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit