Structural information from orientationally selective DEER spectroscopy

Abstract

Double electron-electron resonance (DEER) spectroscopy can determine, from measurement of the dipolar interaction, the distance and orientation between two paramagnetic centres in systems lacking long-range order such as powders or frozen solution samples. In spin systems with considerable anisotropy, the microwave pulses excite only a fraction of the electron paramagnetic resonance (EPR) spectrum and the resulting orientation selection needs to be explicitly taken into account if a meaningful distance and orientation is to be determined. Here, a general method is presented to analyze the dipolar interaction between two paramagnetic spin centres from a series of DEER traces recorded so that different orientations of the spin-spin vector are sampled. Delocalised spin density distributions and spin projection factors (as for example in iron-sulfur clusters), are explicitly included. Application of the analysis to a spin-labelled flavoprotein reductase/reduced iron-sulfur ferredoxin protein complex and a bi-radical with two Cu(ii) ions provides distance and orientation information between the radical centres. In the protein complex this enables the protein-protein binding geometry to be defined. Experimentally, orientationally selective DEER measurements are possible on paramagnetic systems where the resonator bandwidth allows the frequencies of pump and detection pulses to be separated sufficiently to excite enough orientations to define adequately the spin-spin vector.

Direct assignment of EPR spectra to structurally defined iron-sulfur clusters in complex I by double electron–electron resonance

“…Specifically, the simulations (SI Text and ref. (19) for details) require the spin Hamiltonian parameters in Table 1, the coordinates and SPFs for each Fe of each paramagnetic cluster, and the g-axis orientation for each paramagnetic cluster, relative to the molecular frame. The latter quantities are not known precisely, and our procedures to account for them are described next.…”

“…The DEER simulation algorithm has been described previously (19), and its particular application to a set of FeS clusters is summarized in SI Text. In accord with previous work on, for example, NO radicals (12), we chose to fit our data directly in the time domain, to allow simultaneous fitting of both the baseline and the DEER oscillation, and to avoid difficulties associated with the Fourier transformation of DEER traces with large unmodulated contributions.…”

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function

“…Data contained in a single DEER trace will then contain information from which the inter-spin distance cannot be reliably determined. If multiple DEER traces are collected at different magnetic field positions, then the different orientations are sampled and more accurate distance information can be extracted [47]. Orientational selectivity is operative in Cu(II)-Cu(II) and Cu(II)-nitroxide systems [11], and Yang et al [25,26] has recently outlined the methods required to determine if orientational selectivity is present and how to analyse data if it is so.…”

DEER-Stitch: Combining three- and four-pulse DEER measurements for high sensitivity, deadtime free data

“…It has also been applied to a range of other radical systems and at different EPR frequencies (e.g. Q-band and W-band) to measure both distances and relative orientations between spin centres [14][15][16][17][18][19][20][21][22][23][24][25][26][27].…”

“…Nitroxides have a small g-anisotropy and an 14 N hyperfine splitting which makes them suitable for DEER measurements at X-band frequencies ($9.5 GHz) as mw pulses of ca. 12-32 ns excite a large fraction of the spins, the EPR spectrum is wide enough that pump and detection spins can be separately excited, and orientation selection effects are generally small or negligible.…”