Double Resonance Modulation Research Articles

Electron-nuclear double resonance spectroscopy (and electron spin-echo envelope modulation spectroscopy) in bioinorganic chemistry.

This perspective discusses the ways that advanced paramagnetic resonance techniques, namely electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) spectroscopies, can help us understand how metal ions function in biological systems.

Two-dimensional ENDOR-ESEEM correlation spectroscopy

A two-dimensional (2D) experiment that correlates electron-nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) frequencies, useful for unraveling and assigning ENDOR and ESEEM spectra from different paramagnetic centers with overlapping EPR spectra, is presented. The pulse sequence employed is similar to the Davies ENDOR experiment with the exception that the two-pulse echo detection is replaced by a stimulated echo detection in order to enhance the resolution in the ESEEM dimension. The two-dimensional data set is acquired by measuring the ENDOR spectrum as a function of the time interval T between the last two microwave pulses of the stimulated echo detection scheme. This produces a series of ENDOR spectra with amplitudes that are modulated with T. Fourier transformation (FT) with respect to T then generates a 2D spectrum with cross peaks connecting spectral lines of the ESEEM and ENDOR spectra that belong to the same paramagnetic center. Projections along the vertical and horizontal axes give the three-pulse FT-ESEEM and ENDOR spectra, respectively. The feasibility of the experiment was tested by simulating 2D ENDOR-ESEEM correlation spectra of a system consisting of an electron spin (S = (1/2)) coupled to two nuclei (I(1) = I(2) = (1/2)), taking into account experimental conditions such as pulse durations and off-resonance irradiation frequencies. The experiment is demonstrated on a single crystal of Cu(2+) doped l-histidine (Cu-His), containing two symmetrically related Cu(2+) sites that at an arbitrary orientation exhibit overlapping ESEEM and ENDOR spectra. While the ESEEM spectrum is relatively simple and arises primarily from one weakly coupled (14)N, the ENDOR spectrum is very crowded due to contributions from two nonequivalent nitrogens, two chlorides, and a relatively large number of protons. The simple ESEEM projection of the 2D ENDOR-ESEEM correlation spectrum is then used to disentangle the ENDOR spectrum and resolve two sets of lines corresponding to the different sites. Copyright 2000 Academic Press.

Electron Nuclear Quadruple Resonance for Assignment of Overlapping Spectra

Multiple resonance methods are important tools in EPR for revealing the network of hyperfine levels of free radicals and paramagnetic centers. The variations of electron nuclear double resonance (ENDOR) or electron spin-echo envelope modulation (ESEEM) techniques help to correlate nuclear frequencies with each other. These methods have limited utility when there is extensive overlap or suspected overlap in the EPR spectrum between different species or different orientations. In the ENDOR spectrum, overlap and second-order shifts of lines also leads to ambiguity in assignment and interpretation. A new electron nuclear multiple resonance method is presented here that is based on population transfer ENDOR. It is a quadruple resonance method that correlates ENDOR lines and reveals the network of hyperfine levels in samples with unoriented paramagnetic species and in samples with overlapping EPR or ENDOR lines.

Oxo-vanadium as a spin probe for the investigation of the metal coordination environment of imidazole glycerol phosphate dehydratase

Imidazole glycerol phosphate dehydratase (IGPD) catalyses the dehydration of imidazole glycerol phosphate to imidazole acetol phosphate, an important late step in the biosynthesis of histidine. IGPD, isolated as a low molecular weight and inactive apo-form, assembles with specific divalent metal cations to form a catalytically active high molecular weight metalloenzyme. Oxo-vanadium ions also assemble the protein into, apparently, the same high molecular weight form but, uniquely, yield a protein without catalytic activity. The VO 2+ derivative of IGPD has been investigated by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopy. The spin Hamiltonian parameters indicate the presence of multiple 14N nuclei in the inner coordination sphere of VO 2+ which is corroborated by ENDOR and ESEEM spectra showing resonances attributable to interactions with 14N nuclei. The isotropic superhyperfine coupling component of about 7 MHz determined by ENDOR is consistent with a nitrogen of coordinated histidine imidazole(s). The ESEEM Fourier-transform spectra further support the notion that the VO 2+ substituted enzyme contains inner-sphere nitrogen ligands. The isotropic and anisotropic 14N superhyperfine coupling components are similar to those reported for other equatorially coordinated enzymatic histidine imidazole systems. ESEEM resonances from axial 14N ligands are discussed.

Microwave and Millimeterwave Spectrum of 1,1-Difluoroethylene and the Vibrational Mode ν10 from FT-IR Spectroscopy

Abstract Pure rotational spectra of 1,1-Difluoroethylene (1,1-DFE) in the vibrational ground state (GS) and in excited vibrational states (EVSs) have been investigated by double resonance modulation (DRM) microwave spectroscopy in the range from 12 GHz to 40 GHz, and by millimeterwave spectroscopy in the range from 90 to 260 GHz. In addition to the GS spectrum, rotational transitions have been observed for the EVSs ν10 = 1, and 2, ν12 = 1, ν6 = 1, ν11 = 1, ν4 = 1, and ν9 = 1. Along with other IR bands up to 1400cm-1 , the fundamental vibrational band ν10 = 1 ← 0 has been observed by FT-IR spectroscopy and has been analysed to yield ν10 0 = 436.8851 cm-1. Rotational constants and quartic distortion constants were deduced for all observed states. In addition, sextic distortion constants were obtained for the GS and for the EVSs ν10 = 1, 2, ν6 = 1, and ν12 = 1 by computer fits of combined microwave and millimeterwave data to Watson's A-reduced Hamiltonian in Ir representation. The S-reduced Hamiltonian in IIIr representation may be considered more appropriate for the calculation or the fitting of the transitions because 1,1-DFE is a near oblate asymmetric top. Thus, for comparison, results are given for both the A- and the S-reduction.

Two-Dimensional ESEEM Study of VO2+ Complexes with Imidazole and Histidine: Histidine Is a Polydentante Ligand

The oxovanadium cation, VO{sup 2+}, has been successfully employed as a spin probe in electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) studies of metalloprotein active sites. Here we demonstrate that the complex of VO{sup 2+} with histidine has been misinterpreted and that the dominant ESEEM pattern is produced by the {alpha}-amino group. Nevertheless, we show that histidine side chain ligands can be identified from either nitrogen hyperfine or quadrupole couplings. The application of 2D ESEEM to the [{sup 15}N]imidazole complex shows, in the same spectrum, both the coordinated and the remote ring nitrogens with very different isotropic hyperfine couplings. In the case of histidine ligation, equatorial coordination by both imine and amine nitrogen is demonstrated. The reproducible differences between both the hyperfine and the quadrupole constants of the amine and imine nitrogens are reflected in the shape and position of the lines in the 2D spectra and allow them to be distinguished in proteins. With both imine and amine coordination, the VO{sup 2+}/histidine complex cannot be considered an adequate model for coordination by the histidine side chain in proteins. The imidazole, as a monodentante ligand, is better for this role. 16 refs., 3 figs.

Site-directed mutagenesis of conserved histidines in the helix VIII domain of PsaB impairs assembly of the photosystem I reaction center without altering spectroscopic characteristics of P700.

The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P700, A0, and A1. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P700 reaction center chlorophyll(s) and the initial electron acceptor, A0. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P700+. In all cases we determine that spectroscopic characteristics of P700+ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.

Linear and quadratic electrooptic effects in symmetric and asymmetric quantum-well structures

In the first part of the paper, the changes in the complex index of refraction of quantum-well structures proportional to the applied dc electric field (F) and its square are obtained from stationary perturbation theory. Expressions are obtained for the linear and quadratic changes in index and absorption coefficients of asymmetric and symmetric quantum well structures. The triple resonance term does not contribute to the quadratic Kerr effect for a symmetrical well in an expansion about F=0. In the second part of the paper, these results are applied to several types of modulators. First we consider two kinds of silicon/silicon-germanium high-barrier asymmetric quantum well structures at 1.55 /spl mu/m. For the step well, the dominant triple resonant term of the quadratic Kerr effect adds to the linear electrooptic effect (LEG) to yield an intensity extinction ratio of 15 dB in switching from F=+8 V//spl mu/m to -8 V//spl mu/m and a very small insertion loss of 0.04 dB, for a modulator length of 200 pm and for narrow linewidths (FWHM=7.6 meV). For the case of asymmetric barriers confining silicon wells, a comparable insertion loss is obtained but with an extinction ratio of 44 dB for FWHM=20 meV, a clearly superior performance. For symmetrical silicon-based wells, the double-resonance modulation, though small at /spl lambda/=1.55 /spl mu/m, is appreciable at /spl lambda/=10.6 /spl mu/m. For the Kerr effect in symmetrical gallium arsenide wells at /spl lambda/=10.6 /spl mu/m reported by Sa'ar et al., we show that it is the double-resonance term which yields the six order-of-magnitude enhancement with respect to bulk and that an even larger enhancement of the Kerr effect can be obtained in an asymmetric GaAs structure together with the LEO. >

ESEEM and ENDOR studies of the Rieske iron-sulphur protein in bovine heart mitochondrial membranes

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) were applied to the respiratory-chain iron-sulphur clusters in natural bovine heart mitochondrial membranes. By using specific reduction, signals were observed from the Complex III Rieske [2Fe-2S] cluster. In ENDOR, 1H hyperfine couplings in the range 0.5–7 MHz were observed. In ESEEM, modulations were obtained which were assigned to two 14N nuclei of directly-coupled imidazole ligands. The ESEEM spectra are similar to previous observations on purified iron-sulphur proteins of this type, in which the iron-sulphur cluster is coordinated by two cysteine and two histidine ligands. They confirm that the coordination of the cluster in the purified proteins, with two cysteinyl sulphur and two histidine nitrogens, is unchanged from its natural mitochondrial membrane environment. In order to investigate the possible interaction of the membrane-bound Rieske protein with quinones, measurements were conducted on membranes preincubated with 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT), and in the pH range 6–7.5. No significant changes were detected, either in the proton hyperfine couplings as detected by ENDOR, or in the nitrogen couplings to the histidines as detected by ESEEM.

ENDOR and ESEEM studies of cytochrome c oxidase: evidence for exchangeable protons at the CuA site.

Electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopies were used to study whether protons in the immediate protein environment around CuA in cytochrome c oxidase are susceptible to solvent exchange. The enzyme was incubated in buffered D2O under resting or turnover conditions for 90 min and then frozen to quench the hydrogen/deuterium-exchange process. ENDOR spectra of the deuterated sample were essentially identical to those of control samples. The ESEEM spectra, however, provided a clear indication of the introduction of deuterium into the CuA environment following incubation in buffered D2O. The extent of deuterium incorporation was not affected by enzyme turnover. An analysis of the ESEEM data indicated that water is in reasonably close proximity to the CuA site, but not in the immediate coordination sphere of the metal(s). We estimate a minimum distance of 5.4 A between the CuA center and the protein/water interface. This relatively short surface separation distance is consistent with the role of CuA as the immediate oxidant of cytochrome c in the cytochrome oxidase (Hill, B. C. (1991) J. Biol. Chem. 266, 2219-2226).

ESR, ENDOR and ESEEM studies of Lewis acid site interactions with tetrachloro-1,2-benzoquinone in aluminosilicate catalysts

The interactions of o-chloranyl (tetrachloro-1,2-benzoquinone) with Lewis acid sites in HY, H-mordenite and H-ZSM-5 zeolites and in aluminum oxide have been studied using electron spin resonance, electron nuclear double resonance and electron spin echo envelope modulation spectroscopies. The results are compared to those obtained from paramagnetic complexes generated in a solution of o-quinone and AlCl 3 which serves as a model system. The detected 27Al hyperfine interactions lead to the conclusion that adsorption of o-chloranyl on activated catalysts yields a paramagnetic complex with Al 3+ Lewis acid sites. Hence, o-chloranyl can be used as an indicator for the detection and quantification of such sites.

1H and 2D electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) investigations on γ-irradiated betaine arsenate

Electron spin resonance, electron nuclear double resonance and electron spin echo envelope modulation measurements were performed on γ-irradiated betaine arsenate single crystals. The investigations revealed that two radicals, Ṅ(CH 3) 3 and (CH 3) 3N +ĊHCOO −, are formed by the γ-irradiation at room temperature. The concentration of both radicals decays in time with a time constant of a few days. In deuterated betaine arsenate the protons of the CH 2 group are partially replaced by deuterons. The 14N hyperfine coupling tensor of the Ṅ(CH 3) 3 radical as well as the 1H, 2D hyperfine coupling tensor and the 2D quadrupole tensor of the (CH 3) 3N +ĊHCOO − ((CH 3) 3N +ĊDCOO −) radicals were determined.

Technical Memorandum: Triple modulation technique applicable to microwave spectrometers with cavity gas cell

A triple modulation technique, applicable to microwave cavity spectrometers, is described, which enables Stark, Zeeman, double resonance or cavity modulation in the cavity gas cell to operate at a relatively low frequency, while satisfying the criterion that the microwave crystal detector should operate at a relatively high frequency. This technique is such that the microwave source is frequency double-modulated by means of sweeping signal of frequency f1 and a relatively high-frequency sinusoidal signal of frequency f2 applied to a tuning varactor, while an additional relatively low-frequency modulation such as Stark, Zeeman, double resonance or cavity modulation (frequency f3) is applied in the cavity gas cell. The low-frequency modulation signal (f3), which contains the salient information of the analyte gas, is carried by the high-frequency signal f2, the former being extracted and amplified by a series of filters, demodulators and tuned amplifiers.

High-Resolution Vibrational Spectra of Furazan

Abstract The study by Fourier transform (FT) infrared (IR) spectroscopy of the fundamental vibrational bands v12 and v5 of furazan yields the origins of these bands with a statistical uncertainty of 10-6 cm-1, which leads to an estimated absolute uncertainty of 10-4 cm-1. The values are v°12 = 952.6123 cm -1 and v°5 = 1.005.3536 cm -1. They confirm the values previously deduced from laser/microwave double resonance (LMDR) experiments. Previous results for the molecular constants of the vibrational ground state and of the two vibrationally excited states, as obtained by double resonance modulation (DRM) microwave spectroscopy alone, are confirmed and refined. Advantages brought about through the combination of the DRM microwave and the FT-IR technique are outlined.

Microwave Spectra of Furazan. IV. Rotation Spectra of Vibrationally Excited States of Perdeuterated Furazan

Abstract The pure rotation spectra of molecules in 25 vibrationally excited states of perdeuterated furazan, C2D2N2O, have been studied by double resonance modulation (DRM) microwave spectroscopy. Twelve of these spectra have been correlated, -on the basis of relative intensity measurements under DRM -, with fundamental vibrations as previously established by IR spectroscopy. Rotational parameters for these 12 fundamental levels are reported, and the contributions to the effective rotational constants and to the inertia defect of the ground state of d2 -furazan have been determined for 10 modes of vibration.

The Microwave Spectrum of Isopropyl Iodide: The Iodine Hyperfine Structure and a Centrifugal Distortion Analysis

Abstract The ground stafe microwave spectrum of isopropyl iodide has been investigated by microwave Fourier transform (MWFT) spectroscopy. 212 hyperfine components of 39 mainly a-type transitions have been assigned from the measured spectra in the frequency range between 4 and 30 GHz. With a MWFT double resonance modulation (MWFTDR) spectrometer a weak c-type transition and a "forbidden" ΔJ = 2 line could be observed. The spectra were analysed using a diagonalization procedure of the complete Hamiltonian matrix and a simultaneous least squares fit of the rotational, quartic centrifugal distortion and the iodine quadrupole and spin rotation coupling constants directly to the measured frequencies. A set of 14 constants could be obtained with high accuracy. The rotational constant A and some of the centrifugal distortion constants could be improved using some large higher order quadrupole perturbations and the results of the MWFTDR measurements for the analysis.

A Microwave Fourier Transform Spectrometer in the Region from 4 to 6 GHz with Double Resonance Modulation

Abstract We report on the experimental set up and performance of a microwave Fourier transform spectrometer for the G band from 4 to 6 GHz. The sensitivity, achievable linewidth, and resolution capability is demonstrated by measurements of rotational transitions of various molecules.

The Triply Determined Substitution Structure of 1,3,4-Thiadiazole by DRM Microwave Spectroscopy

Double Resonance Modulation (DRM) microwave spectroscopy has allowed the determination of the complete substitution structure of normal, mono-deuterated and perdeuterated 1,3.4-thiadiazole (N2C2H2S). The shift of the centre of gravity between the normal and the d2-form and the swing of inertia! axes relative to the C2v symmetrical forms in the 2-d1-species have been used to revise the small coordinates of hydrogen and deuterium atoms. Bond lengths (in Å) and bond angles (in degrees), with uncertainties covering all three molecular forms, are found as follows: The results suggest that the shift and the swing of inertial axes between different ‘parent’ forms, which can both be determined from the moments of multiply substituted molecular isotopes, may be used with advantage to refine the position of near-axis atoms.

Nuclear Quadrupole Hyperfine Structure in the Rotational Spectrum of 3-Chloropyridine. An Application of Microwave-Microwave Double Resonance Fourier Transform Spectroscopy

Abstract We redetermined the rotational and the chlorine-35 and nitrogen-14 nuclear quadrupole coupling constants of 3-chloropyridine. The values are A = 5839.5330(12) MHz, B = 1604.1875(6) MHz, and C = 1258.3121 (5) MHz for the rotational constants, and χaa(Cl) = - 72.255(19) MHz, χbb(Cl) = + 38.500(13) MHz, χcc(Cl) = + 33.755(23) MHz and χaa(N) = - 0.009(13) MHz, χbb(N) = - 3.473(10) MHz, χCC(N) = + 3.482(16) MHz for the chlorine-35 and nitrogen-14 nuclear quadrupole coupling constants, respectively. Application of double resonance modulation technique is shown to greatly simplify the assign­ment of hyperfine structure components even of weak rotational transitions.

Micro wave Spectra of Furazan I. The Doubly Determined Substitution Structure

Double Resonance Modulation (DRM) microwave spectroscopy has been used to determine the rotational constants of isotopic forms of normal (C2H2N2O2) and perdeuterated furazan (C2D2N2O). From these data the complete substitution structures have been derived by Kraitchman methods and by the computer program GEOM. The latter procedure fails to give correct results due to the decrease of the moment of inertia about the C2v -axis and the simultaneous interchange of inertial axes in the 18O-3,4-d2-form. The following bondlengths (in Å) and angles (in degrees) are best compatible with the total body of isotopic information: 0 - N = 1.3729 [6] ∢ NON = 111.15 [8] N -C = 1.3032 [13] ∢ ONC = 105.53 [5] C -C =1.4211 [9] ∢ NCC = 108.89 [3] C -H = 1.0763 [5] ∢ CCH = 130.16 [3] Here, the uncertainties cover small differences between normal and d2-furazan as well as discrepancies resulting from different schemes of structure calculation.