Pulsed electron–electron double resonance spectroscopy between a high-spin Mn²⁺ ion and a nitroxide spin label

Abstract

Pulsed Electron-Electron Double Resonance (PELDOR) has attracted considerable attention for biomolecular applications, as it affords precise measurements of distances between pairs of spin labels in the range of 1.5-8 nm. Usually nitroxide moieties incorporated by site-directed spin labelling with cysteine residues are used as spin probes in protein systems. Recently, naturally occurring cofactors and metal ions have also been explored as paramagnetic spin species for such measurements. In this work we investigate the performance of PELDOR between a nitroxide spin label and a high-spin Mn(2+) ion in a synthetic model compound at Q-band (34 GHz) and G-band (180 GHz). We demonstrate that the distances obtained with high-frequency PELDOR are in good agreement with structural predictions. At Q-band frequencies experiments have been performed by probing either the high-spin Mn(2+) ion or the nitroxide spin label. At G-band frequencies we have been able to detect changes in the dipolar oscillation frequency, depending on the pump-probe positions across the g-tensor resolved nitroxide EPR spectrum. These changes result from the restricted mobility of the nitroxide spin label in the model compound. Our results demonstrate that the high-spin Mn(2+) ion can be used for precise distance measurements and open the doors for many biological applications, as naturally occurring Mg(2+) sites can be readily exchanged for Mn(2+).

Distance measurements between manganese(ii) and nitroxide spin-labels by DEER determine a binding site of Mn2+ in the HP92 loop of ribosomal RNA

“…36 Fig . We demonstrate this approach by locating a Mn 2+ binding site in an RNA derived from hairpin 92 of the 23S ribosomal RNA (HP92).…”

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…[12][13][14] The most commonly used spin label is (1-oxyl-2,2,5,5-tetra-methylpyrroline-3-methyl)-methanethiosulfonate (MTSSL), which specifically reacts with the thiol group of cysteine residues. [20][21][22][23] The search for alternative spin labels is an active area of research. [19] Recently, Cu(II) and high spin Mn(II) tags have also been successfully employed.…”

“…The correct determination of the zero-time is particularly important for short inter-spin distances which give rise to high frequency dipolar oscillations. [20] The distance between the center of the tetraphenylporphyrin and the NÀ O midpoint is indicated. [8] The performance of LaserIMD and ReLaserIMD for the model peptide (Figure 1(a)) is compared in Figure 2.…”

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…However, in contrast to Gd 3+ , the ZFS of Mn 2+ spans a much larger range of values. Depending on the ligands, the axial ZFS constant | D | falls into the range from 280 MHz to 6.2 GHz . At microwave frequencies larger than Q‐band, at which the ZFS becomes much smaller than the Zeeman term, the central transition | m S =−1/2>↔| m S =+1/2> is the most intense and narrow component of the Mn 2+ spectrum, whereas the other four transitions are broad and contribute only to the background of the spectrum.…”

“…1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 axial ZFS constant j D j falls into the range from 280 MHz to 6.2 GHz. [113,114] At microwave frequencies larger than Q-band, at which the ZFS becomes much smaller than the Zeeman term, the central transition j m S = À 1/2 > $ j m S = + 1/2 > is the most intense and narrow component of the Mn 2 + spectrum, whereas the other four transitions are broad and contribute only to the background of the spectrum. In analogy to Gd 3 + , the width of the central transition is proportional to the ratio D 2 /(gβ e B 0 ) and narrows as the magnetic field increases.…”