Laser-Induced Magnetic Dipole Spectroscopy

Abstract

Pulse electron paramagnetic resonance measurements of nanometer scale distance distributions have proven highly effective in structural studies. They exploit the magnetic dipole-dipole coupling between spin labels site-specifically attached to macromolecules. The most commonly applied technique is double electron-electron resonance (DEER, also called pulsed electron double resonance (PELDOR)). Here we present the new technique of laser-induced magnetic dipole (LaserIMD) spectroscopy based on optical switching of the dipole-dipole coupling. In a proof of concept experiment on a model peptide, we find, already at a low quantum yield of triplet excitation, the same sensitivity for measuring the distance between a porphyrin and a nitroxide label as in a DEER measurement between two nitroxide labels. On the heme protein cytochrome C, we demonstrate that LaserIMD allows for distance measurements between a heme prosthetic group and a nitroxide label, although the heme triplet state is not directly observable by an electron spin echo.

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…In parallel, combining the nitroxide and the endogenous probe in an orthogonal labelling approach has proven to be very effective since the spectroscopically non-identical labels can be addressed selectively during the PDS experiment. [30] However, no triplet state was observed by EPR spectroscopy, as expected for a low-spin ferric heme. [35][36][37][38] Recently, it has been shown that the RIDME experiment is better suited than DEER for distance measurements between spin active moieties with different spin-lattice relaxation times or species with very broad spectra such as metal ions like low-spin Fe(III).…”

“…[24] One important new development is the demonstration that the triplet state of porphyrin chromophores can be exploited to determine inter-spin distances. [30] Comparison between the two techniques was carried out both at X-band and at Q-band. [25,26] The large electron spin polarization of the photoexcited triplet state, [27,28] and the consequently high sensitivity of the experiment, furthermore allowed light-induced PDS methodology to be applied to a photosynthetic protein, containing an endogenous carotenoid triplet state probe.…”

“…For this purpose we employed an α-helix peptide, used in previous studies, [25,26,30] labeled with a tetra-phenylporphyrin moiety and with the unnatural amino acid TOAC (4-amino-1-oxyl-2,2,6,6-tetra-methylpiperidine-4-carboxylic acid). For this purpose we employed an α-helix peptide, used in previous studies, [25,26,30] labeled with a tetra-phenylporphyrin moiety and with the unnatural amino acid TOAC (4-amino-1-oxyl-2,2,6,6-tetra-methylpiperidine-4-carboxylic acid).…”

“…The absence of symmetry in the complete LaserIMD trace (see the Supporting Information) does not allow the symmetry-based procedure for zero determination to be used in the analysis our experimental data as proposed by Hintze et al [30] For this reason, in a technique we dub ReLaserIMD (Figure 2 right), we employ the same principle as in the 4-pulse DEER scheme, in which a refocused echo detection sequence is utilized, to yield a symmetric zero-time. The correct determination of the zero-time is particularly important for short inter-spin distances which give rise to high frequency dipolar oscillations.…”

“…In the LaserIMD trace several points could be picked as potential zero times in the zone where the change of slope between the baseline and the drop of the first modulation occurs, preventing the procedure being free from bias. Thus while the LaserIMD experiment is free from experimental dead-time due to pulse overlap, [30] there is still a shift in the zero time, which could be considered a zero-time artefact, arising from the finite length of the pulses. This important parameter affects the output of the distance analysis: the different distributions obtained from LaserIMD have their maxima spread over a range of distances of about 0.1 nm, whereas this interval is limited to about 0.01 nm for ReLaserIMD.…”