Abstract
Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling constants and thus the distance between electron spin centers. Up to now, PDS measurements have been mostly applied to spin centers whose g‐anisotropies are moderate and therefore have a negligible effect on the dipolar coupling constants. In contrast, spin centers with large g‐anisotropy yield dipolar coupling constants that depend on the g‐values. In this case, the usual methods of extracting distances from the raw PDS data cannot be applied. Here, the effect of the g‐anisotropy on PDS data is studied in detail on the example of the low‐spin Fe3+ ion. First, this effect is described theoretically, using the work of Bedilo and Maryasov (Appl. Magn. Reson. 2006, 30, 683–702) as a basis. Then, two known Fe3+/nitroxide compounds and one new Fe3+/trityl compound were synthesized and PDS measurements were carried out on them using a method called relaxation induced dipolar modulation enhancement (RIDME). Based on the theoretical results, a RIDME data analysis procedure was developed, which facilitated the extraction of the inter‐spin distance and the orientation of the inter‐spin vector relative to the Fe3+ g‐tensor frame from the RIDME data. The accuracy of the determined distances and orientations was confirmed by comparison with MD simulations. This method can thus be applied to the highly relevant class of metalloproteins with, for example, low‐spin Fe3+ ions.
Highlights
Pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS), which includes techniques such as pulsed electron–electron double resonance (PELDOR or DEER),[1,2] double quantum coherence EPR (DQC),[3] single-frequency technique for refocusing dipolar couplings (SIFTER),[4] and relaxation induced dipolar modulation enhancement (RIDME),[5,6] is a valuable method for determining biomolecular structures and their conformational changes during function.[7]
In order to confirm the LS-state of the Fe3+ ions in 1·Im2, 2·Im2 and 1T·Im2, Xband cw-EPR spectra of these compounds were measured at 15 K (Figures 4 and S2)
The obtained spectra do show the characteristic signal of the LS Fe3+ ion, which is overlaid with the sharp saturated signal of the nitroxide or trityl radicals
Summary
The first application of this theory for the analysis of RIDME data was reported by Astashkin et al.[29] There, the RIDME spectrum of the LS Fe3+ /flavin spin pair was simulated using a modified equation for the dipolar coupling constant, which provided estimates of the inter-spin distance and two angles that determine the relative orientation of the Fe3+ g-tensor with respect to the distance vector This analysis was done only in a semi-quantitative way, because usage of the four-pulse RIDME sequence lead to time traces with significant dead time and the SNR was rather low.
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