High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

• DOI: 10.1073/pnas.0605851103
• Publication Date: 2006-08-29T02:10:40Z
• Authors: V. P. Denysenkov; T. F. Prisner; J. Stubbe; M. Bennati
• Keywords: Ribonucleotide reductase; Pulsed EPR; Site-directed spin labeling

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, is site conversion nucleoside diphosphates to 2'-deoxynucleoside diphosphates. It has been proposed that function tyrosyl in generate a transient thiyl (C439.) over distance 35 A, which turn initiates reduction process. EPR measurements provide tool with study mechanism initiation class RNRs. These types experiments at low magnetic fields frequencies (0.3 T, 9 GHz) give insight into interradical distances populations. We present pulsed electron-electron double resonance (PELDOR) experiment high frequency (180-GHz electron Larmor frequency) detects dipolar interaction between Y.s each protomer RNR from Escherichia coli. observe correlation orientation-dependent their resolved g-tensors. This information allowed us define relative orientation radicals embedded active homodimeric protein solution. demonstrates high-field PELDOR spectroscopy powerful assembly proteins contain multiple paramagnetic centers.