Cross‐Polarization Electron‐Nuclear Double Resonance Spectroscopy

Abstract

Magnetic nuclei in the proximity of a paramagnetic center can be polarized through electron-nuclear cross-polarization and detected in electron-nuclear double resonance (ENDOR) spectroscopy. This principle is demonstrated in a single-crystal model sample as well as on a protein, the β2 subunit of E.coli ribonucleotide reductase (RNR), which contains an essential tyrosyl radical. ENDOR is a fundamental technique to detect magnetic nuclei coupled to paramagnetic centers. It is widely employed in biological and materials sciences. Despite its utility, its sensitivity in real samples is about one to two orders of magnitude lower than conventional electron paramagnetic resonance, thus restricting its application potential. Herein, we report the performance of a recently introduced concept to polarize nuclear spins and detect their ENDOR spectrum, which is based on electron-nuclear cross polarization (eNCP). A single-crystal study permits us to disentangle eNCP conditions and CP-ENDOR intensities, providing the experimental foundation in agreement with the theoretical prediction. The CP-ENDOR performance on a real protein sample is best demonstrated with the spectra of the essential tyrosyl radical in the β2 subunit of E.coli RNR.

Properties of Site-Specifically Incorporated 3-Aminotyrosine in Proteins To Study Redox-Active Tyrosines: Escherichia coli Ribonucleotide Reductase as a Paradigm

“…An alternative method to obtain the angle θ is the use of orientation-selected HF ENDOR spectroscopy, however the low radical yield of D 6 -NH 2 Y 731 • unfortunately has so far prevented these experiments. We are in the process to introduce CP-edited ENDOR 71 for 2 H nuclei, which will potentially facilitate the analysis of 2 H-HF ENDOR spectra. Preliminary 94 GHz 2 H CP-ENDOR data (not shown) on H 6 -ND 2 Y 731 •-α2 seem entirely consistent with the present analysis.…”

Cross-polarisation ENDOR for spin-1 deuterium nuclei

“…Again, we suggest that the absence of the negative peak is due to a cancellation resulting from sample inhomogeneities, which are not considered in the analytical treatment, as we neglect any off-resonance effects. Recent analysis of powder patterns [33,37] indicated that negative peaks disappear in the presence of such inhomogeneities. Nevertheless, the overall agreement between different theoretical predictions and the experimental data is striking and not obvious, as the analytical prediction of intensities (Table 3) does not explicitly calculate a time evolution of the density operator during the spin lock, whereas the numerical simulation does.…”

Pulsed Electron Decoupling and Strategies for Time Domain Dynamic Nuclear Polarization with Magic Angle Spinning

“…47,48 Importantly, all three of these time domain DNP transfers can be implemented with chirped frequency microwave irradiation, rather than square, hard pulses. Such frequency-swept strategies are not only robust to microwave field inhomogeneity but also can be readily implemented with existing frequency-agile gyrotrons.…”

“…eNCP (Figure 3c) is another promising time domain DNP experiment 47,48 and could be implemented with readily available frequency-agile gyrotron microwave sources. Large hyperfine couplings present in a direct-transfer eNCP experiment result in differing effective fields between the and spin states of the nuclei and electrons The matching condition for eNCP is given by eq 6, where ν Sα eff and ν Sβ eff are the effective fields of the and electrons, respectively, and ν Iα eff and ν Iβ eff are the effective fields of the and nuclei, respectively.…”

“…Large hyperfine couplings present in a direct-transfer eNCP experiment result in differing effective fields between the and spin states of the nuclei and electrons The matching condition for eNCP is given by eq 6, where ν Sα eff and ν Sβ eff are the effective fields of the and electrons, respectively, and ν Iα eff and ν Iβ eff are the effective fields of the and nuclei, respectively. The expressions for calculating these effective fields have been shown previously in the literature and appear in eq 7(47,48)…”