High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase.
Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y.) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2'-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439.) in R1 over a distance of 35 A, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y.s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.
Denysenkov
V P
VP
Institute for Physical and Theoretical Chemistry and Center for Biomolecular Magnetic Resonance, J. W. Goethe University, 60438 Frankfurt am Main, Germany.
Prisner
T F
TF
Stubbe
J
J
Bennati
M
M
D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D005609:Free Radicals / Q000032:analysis / Q000737:chemistry*; D007700:Kinetics; D008956:Models, Chemical; D021122:Protein Subunits / Q000737:chemistry; D012264:Ribonucleotide Reductases / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification / Q000378:metabolism; D012996:Solutions / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D029968:Escherichia coli Proteins; D005609:Free Radicals; D021122:Protein Subunits; D012996:Solutions; D014443:Tyrosine; D012264:Ribonucleotide Reductases