Characterization of Y122F R2 of Escherichia coli ribonucleotide reductase by time-resolved physical biochemical methods and X-ray crystallography.
Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the conversion of ribonucleotides to deoxyribonucleotides. It is composed of two homodimeric subunits, R1 and R2. R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process. The in vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physical biochemical methods. An intermediate, Fe3+/Fe4+ cluster (intermediate X), has been identified that is thought to be directly involved in the oxidation of Y122 to the tyrosyl radical (*Y122). An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate intermediate X at sufficient levels that it can be studied using a variety of spectroscopic methods. The details of the reconstitution of the apo and diferrous forms of Y122F R2 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic resonance, and Mössbauer spectroscopies. In addition the structure of this mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 A resolution has been determined. These studies suggest that Y122F R2 is an appropriate model for the examination of intermediate X in the assembly process. Studies with two mutants, Y356F and double mutant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR.
Tong
W
W
Departments of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Burdi
D
D
Riggs-Gelasco
P
P
Chen
S
S
Edmondson
D
D
Huynh
B H
BH
Stubbe
J
J
Han
S
S
Arvai
A
A
Tainer
J
J
D019943:Amino Acid Substitution / Q000235:genetics; D001059:Apoproteins / Q000737:chemistry; D003080:Cold Temperature; D003432:Cross-Linking Reagents; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005290:Ferric Compounds / Q000737:chemistry*; D005609:Free Radicals; D005615:Freezing; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010100:Oxygen; D010649:Phenylalanine / Q000235:genetics; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D015204:Spectroscopy, Mossbauer; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
D001059:Apoproteins; D003432:Cross-Linking Reagents; D005290:Ferric Compounds; D005609:Free Radicals; D014443:Tyrosine; D010649:Phenylalanine; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; D010100:Oxygen