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

Abstract

Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) 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 (C439⅐) in R1 over a distance of 35 Å, 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 Y⅐s 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.double electron-electron resonance ͉ distance measurements R ibonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Class I RNR from Escherichia coli is composed of two homodimeric subunits (R1 and R2) that are thought to form a 1:1 complex (1). The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2Ј-deoxynucleoside diphosphates. The chemistry of nucleotide reduction is moderately well understood (2), and structures of R1 (3) and R2 (4, 5), as well as a recent structure of the R1:R2 holocomplex (6), are available. A major unresolved issue in this class of enzymes is the mechanism of radical initiation (7): How does the tyrosyl radical in R2 generate a transient thiyl radical in R1 over a distance of 35 Å? The current proposal for the radical propagation pathway is based on a docking model of R1 and R2 and involves aromatic amino acid residues (3,8,9). Evidence in support of the long distance and the docking model has been recently provided by pulsed EPR distance measurements (10). These experiments have detected the distance between the Y⅐ in R2 and a nitrogencentered radical in the active site of R1, providing structural information on the R1:R2 active complex in the presence of the substrate and the allosteric effector. The results provided the impetus to further explore the capability of the method at high magnetic fields.The pulsed electron-electron double resonance (PELDOR) experiment detects weak dipolar interactions between radicals and is based on a two-frequency pulse sequence (11, 12). One frequency is requ...

Conformational flexibility of nitroxide biradicals determined by X-band PELDOR experiments

“…Especially at high magnetic field values, where the g-tensor anisotropy of the radicals is usually resolved, such orientation selectivity can be readily achieved [7] as has been demonstrated for two tyrosyl radicals in a ribonucleotide reductase dimer at G-band frequencies (180 GHz / 6.5 T). In this case a strong orientation dependence of the experimentally observed dipolar oscillation frequency could be observed permitting to additionally obtain the relative orientation of the tyrosyl radicals with respect to the R vector [8]. Such orientation selection and angular correlation effects have already been observed in nitroxide biradicals with polyphenyleneethynylene linkers at X-band (9 GHz / 0.3 T), S-band (3 GHz/ 0.1 T) [6,9] and W-band (95 GHz / 3.4 T) [10] frequencies.…”

Advanced EPR Methods for Studying Conformational Dynamics of Nucleic Acids

“…Intrinsic paramagnetic cofactors, as amino acid radicals, metal ions, and iron sulfur centers exhibit a much higher accuracy in the distance determination, because they are usually rigidly incorporated into the protein environment (Becker & Saxena, 2005;Bennati et al, 2003;Biglino, Schmidt, Reijerse, & Lubitz, 2006;Denysenkov, Prisner, Stubbe, & Bennati, 2006;Kay, Elsässer, Bittl, Farrell, & Thorpe, 2006;Roessler et al, 2010;Van Amsterdam, Ubbink, Canters, & Huber, 2003). However, additional effort has to be undertaken to extract the distance information for such centers.…”

“…Because the orientation between both paramagnetic centers cannot be assumed to be randomly distributed, Tikhonov regularization (Chiang et al, 2005;Jeschke et al, 2006Jeschke et al, , 2002 cannot be used to obtain the distance distribution function directly from the PELDOR time trace. Instead, more elaborate simulation methods have to be used to disentangle distances and angular information (Denysenkov et al, 2006;Margraf, Bode, Marko, Schiemann, & Prisner, 2007;Marko et al, 2010Marko et al, , 2009Polyhach, Godt, Bauer, & Jeschke, 2007;Schiemann, Cekan, Margraf, Prisner, & Sigurdsson, 2009). …”

A dual-mode microwave resonator for double electron–electron spin resonance spectroscopy at W-band microwave frequencies

“…Among the most important is the pulsed electron-electron double resonance technique (PELDOR, also called DEER), which allows measurement of long range distances (2-8 nm) between two paramagnetic species in macromolecules [1][2][3][4][5][6]. At high magnetic fields, PELDOR experiments can also deliver information about the relative orientations of paramagnetic species [7][8][9][10][11][12][13]. This is a valuable information to study conformational changes of macromolecules or assemblies of protein complexes in which paramagnetic centers are rigidly embedded.…”