Effects in 94 GHz Orientation-Selected PELDOR on a Rigid Pair of Radicals with Non-Collinear Axes

Abstract

For aromatic organic radicals, pulsed electron-electron double resonance (PELDOR) experiments at high magnetic fields provide information not only about the distance between the paramagnetic species but also about their relative orientation. However, the three-dimensional biradical structure is encoded in a complex pattern of orientation-selected PELDOR traces and the execution of the experiment is generally aggravated by constraints posed by the available hardware and the intrinsically low modulation depth observed. We present a 94 GHz PELDOR experiment performed with a commercial spectrometer and probe heads that permit separation of pump and detection frequencies up to 150 MHz. The setup is employed to examine the orientation selections on a general case of rigid biradicals with non-collinear g axes. The interacting radicals, a tyrosyl radical (Y 122 Á) located in the b2 subunit and an 3-aminotyrosyl radical (NH 2 Y 731 Á) located in the a2 subunit, are generated by Escherichia coli ribonucleotide reductase with a 3-aminotyrosine (NH 2 Y) site specifically incorporated into a2 in the presence of cytidine 5 0-diphosphate and adenosine 5 0-triphosphate. The experimental designs as well as some characteristic features of the observed modulation pattern are discussed.

Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731

“…4A ). 25 , 62 To gain insight into the location of NH 2 Y 731 ˙/R 411 A-α2, three sets of PELDOR experiments were recorded using broadband excitation with a high-power Q-band set up at different excitation positions in the EPR line 63 – 66 (see Fig. 4B and S8 † ).…”

PELDOR analysis of enzyme-induced structural changes in damaged DNA duplexes

“…A detailed study of the orientational selectivity at X-band has been performed for nitroxyl biradicals, [62][63][64][65] for Cu(II) ions and nitroxyl labels in porphyrin 61,64 and in spin-labeled proteins of tetramer KcsA. 66 The orientational selectivity is most notable in high frequency PELDOR spectroscopy, [67][68][69][70][71][72][73] due to the high g-factor resolution.…”

Orientation selection in distance measurements between nitroxide spin labels at 94 GHz EPR with variable dual frequency irradiation

“…A commercial W-band resonator (Bruker TeraFlex) was used that allows for a maximal separation of pump and detection frequencies of 56 MHz at 400 mW of available microwave power (Bruker Power Upgrade 2). 12 For the RNA duplex (Fig. 2b, left), the orientation selection at 94 GHz shows clear deformations of the Pake patterns, with the parallel component of the dipolar tensor (D J ) appearing at resonances close to g z in the EPR spectrum and maximal modulation depth observed between g x and g y .…”

“…This is not only due to the variety of possible conformations that some labels can adopt within the bio-macromolecule, but moreover, to the difficulties related to the performance of a two frequency pump-probe pulsed EPR experiment in the millimeter-wave range, as discussed in several recent papers. 7, [9][10][11][12][13][14][15] This bottleneck has prevented an extended examination of orientation selection at high frequencies in contrast to the method at low EPR frequencies (9 GHz). [16][17][18][19] Particularly, recent analysis of orientation selection at low frequencies revealed that the problem of reconstructing bi-radical structures based on triples of Euler angles is non-trivial due to ambiguities in the solutions.…”