Pulsed Electron-Nuclear Double Resonance (ENDOR) at 140 GHz

Bennati, Marina; Farrar, Christian T.; Bryant, Jeff; Inati, Souheil; Weis, V.; Gerfen, Gary J.; Riggs-Gelasco, Pamela J.; Stubbe, JoAnne; Griffin, Robert G.

doi:10.1006/jmre.1999.1727

Cited by 106 publications

(128 citation statements)

References 25 publications

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“…BDPA is a stable organic radical with a small g-anisotropy ( g x-z ≈ 0.0005) [30]. The spin density distribution is alternating, leading to two distinct, almost isotropic hyperfine couplings.…”

Section: Results and Discussion Epr And Davies Endor Of 1 H-bdpamentioning

confidence: 99%

Cross-polarisation edited ENDOR

et al. 2013

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Electron-nuclear double resonance (ENDOR) is a fundamental technique in electron paramagnetic resonance (EPR) spectroscopy that directly detects hyperfine transitions of nuclei coupled to a paramagnetic centre. Despite its wide use, spinsensitivity and restricted spectral resolution in powder samples pose limitations of this technique in modern application fields of EPR. In this contribution, we examine the performance of an ENDOR pulse sequence that utilises a preparation scheme different from conventional Davies ENDOR. The scheme is based on electron-nuclear cross-polarisation (eNCP), which requires concomitant microwave (MW) and radio-frequency (RF) irradiation satisfying specific matching conditions between the MW and RF offsets and the hyperfine coupling. Changes in nuclear polarisation generated during eNCP can be detected via a conventional ENDOR read-out sequence consisting of an RF π -pulse followed by EPR-spin echo detection. Using 1 H-BDPA as a standard sample, we first examine the CP matching conditions by monitoring the depolarisation of the electron spin magnetisation. Subsequently, so-called CP-edited ENDOR spectra for different matching conditions are reported and analysed based on the provided theoretical description of the time evolution of the spin density matrix during the experiment. The results demonstrate that CP-edited ENDOR provides additional information with respect to the sign of the hyperfine couplings. Furthermore, the sequence is less sensitive to nuclear saturation effects encountered in conventional ENDOR.

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Section: Results and Discussion Epr And Davies Endor Of 1 H-bdpamentioning

confidence: 99%

Cross-polarisation edited ENDOR

et al. 2013

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“…The achievable orientation selection of ENDOR spectra for tyrosine radicals in frozen solution samples has been demonstrated before (53). High-field ENDOR on crystalline samples will provide complete orientational information of all hfc tensors, in particular for the H-bond proton.…”

Section: Discussionmentioning

confidence: 99%

Photosystem II single crystals studied by EPR spectroscopy at 94 GHz: The tyrosine radical Y\documentclass[12pt]{minimal}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\setlength{\oddsidemargin}{-69pt}\begin{document}\begin{equation}{\mathrm{_{D}^{{\bullet}}}}\end{equation}\end{document}

Hofbauer

Zouni

Bittl

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Electron paramagnetic resonance (EPR) spectroscopy at 94 GHz is used to study the dark-stable tyrosine radical Y D• in single crystals of photosystem II core complexes (cc) isolated from the thermophilic cyanobacterium Synechococcus elongatus. These complexes contain at least 17 subunits, including the water-oxidizing complex (WOC), and 32 chlorophyll a molecules͞PS II; they are active in light-induced electron transfer and water oxidation. I n oxygenic photosynthesis, two photosystems (PS I and PS II) function in sequence to convert light into energy-rich chemical compounds (1, 2). PS I uses energy from the absorption of a photon to reduce NADP ϩ to NADPH, which is required for CO 2 reduction. The electrons for this process are donated by PS II. On light excitation of PS II, an electron is transferred from the primary donor P 680 , a chlorophyll species, via an intermediate pheophytin Pheo a to the plastoquinone acceptors Q A and Q B . Two sequential univalent redox steps and concomitant protonation events lead to plastohydroquinol Q B H 2 , which leaves PS II and provides electrons to PS I via the cytochrome b 6 f complex (for review, see ref.2). The photooxidized cation radical P 680•ϩ has the highest oxidation potential of all cofactors known in nature (Նϩ1.1 V), which is sufficient for water oxidation. P 680•ϩ extracts an electron from a redox active tyrosine Y Z . The intermediate tyrosine radical Y Z• , in turn, oxidizes the water-oxidizing complex (WOC), a tetranuclear manganese cluster. The WOC passes through a cycle of four one-electron oxidation steps in which water is oxidized and protons and O 2 are released (for reviews, see refs. 3-7). The exact water-splitting mechanism is still unknown.The cofactors involved in the electron transfer chain of PS II are bound to two protein subunits, D 1 and D 2 . From amino acid sequence homology (8-10), two-dimensional electron crystallography (11), and computer modeling (12), D 1 and D 2 are assumed to be arranged analogously to the L and M subunits in the reaction center of purple bacteria. This analogy has been supported recently by x-ray crystallographic studies of the PS II single crystals (13). Whereas Y Z in D 1 connects P 680•ϩ to the WOC in the electron transfer chain, the homologous Y D in D 2 does not seem to take part in the charge separation process. However, Y D is also coupled to the WOC and, under illumination, forms a dark-stable radical Y D• (Fig. 1). The functional role of Y D is not understood in detail; it may be necessary for assembly of the PS II complex (14, 15). Recent results also suggest that it may play a role in preventing photoinhibition during activation of the PS II complex (16).In the light-induced single electron transfer process and in the water-splitting cycle, various paramagnetic species are formed (17) that have been studied by conventional X-band (9 GHz) electron paramagnetic resonance (EPR) techniques during the last decade. Most of these species can be observed only in the freeze-trapped state in frozen PS II solutions. ...

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“…For simplicity, no additional inhomogeneous broadening function in the EPR spectrum was accounted in the angle selection. This procedure had been used in the past for the computation of orientation-selected electron nuclear double resonance spectra of tyrosyl radicals at similar fields (31,41) and had led to satisfactory results. The modulation depth was computed as the fraction of spins belonging to a spin pair with respect to the total spectral intensity at the field of detection (24).…”

Section: Methodsmentioning

confidence: 99%

High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

Denysenkov

Prisner

Stubbe

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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142

117

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Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439⅐) in R1 over a distance of 35 Å, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y⅐s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.double electron-electron resonance ͉ distance measurements R ibonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Class I RNR from Escherichia coli is composed of two homodimeric subunits (R1 and R2) that are thought to form a 1:1 complex (1). The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2Ј-deoxynucleoside diphosphates. The chemistry of nucleotide reduction is moderately well understood (2), and structures of R1 (3) and R2 (4, 5), as well as a recent structure of the R1:R2 holocomplex (6), are available. A major unresolved issue in this class of enzymes is the mechanism of radical initiation (7): How does the tyrosyl radical in R2 generate a transient thiyl radical in R1 over a distance of 35 Å? The current proposal for the radical propagation pathway is based on a docking model of R1 and R2 and involves aromatic amino acid residues (3,8,9). Evidence in support of the long distance and the docking model has been recently provided by pulsed EPR distance measurements (10). These experiments have detected the distance between the Y⅐ in R2 and a nitrogencentered radical in the active site of R1, providing structural information on the R1:R2 active complex in the presence of the substrate and the allosteric effector. The results provided the impetus to further explore the capability of the method at high magnetic fields.The pulsed electron-electron double resonance (PELDOR) experiment detects weak dipolar interactions between radicals and is based on a two-frequency pulse sequence (11, 12). One frequency is requ...

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Pulsed Electron-Nuclear Double Resonance (ENDOR) at 140 GHz

Cited by 106 publications

References 25 publications

Cross-polarisation edited ENDOR

Cross-polarisation edited ENDOR

High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

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