Electron Nuclear Double Resonance Signals Research Articles

An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5.

Previous Mössbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic [4Fe-4S]2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mössbauer and ENDOR properties. In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms. In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe. Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry. The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center. The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters. The observation of 57Fe ENDOR signals from two types of iron site ([A1c] approximately 30-34 MHz; [A2c] approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by [A1c] confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.

17O electron nuclear double resonance characterization of substrate binding to the [4Fe-4S]1+ cluster of reduced active aconitase.

To characterize the binding of substrate to aconitase, we have made 17O electron nuclear double resonance (ENDOR) measurements on reduced active ([4Fe-4S]1+) beef heart aconitase, both in H216O and H217O, in the presence of substrate and the inhibitors, tricarballylate, trans-aconitate, and 1-hydroxy-2-nitro-1, 3-propanedicarboxylate, referred to here as nitroisocitrate; the hydroxyl of the latter also was isotypically labeled with 17O. The hydroxyl oxygen of citrate and isocitrate is exchanged with solvent water by aconitase, but the hydroxyl of nitroisocitrate is not. Thus, the isotopic composition of nitroisocitrate can be chemically controlled, allowing direct identification of any 17O ENDOR signal associated with it. 17O ENDOR signals were observed from Hx17O (mean = 1 or 2) bound to the [4Fe-4S]1+ cluster in samples prepared with trans-aconitate and unlabeled nitroisocitrate. 17O-Labeled nitroisocitrate in H216O bound to the cluster showed a signal from the 17OH group; in H217O it showed 17O ENDOR resonances due to both Hx17O and 17OH of substrate. This result demonstrates that the cluster participates in substrate binding and can simultaneously coordinate the hydroxyl of a substrate (or analogue) and water (or hydroxyl). The sample with citrate in H217O showed only the Hx17O signal, although aconitase exchanges the hydroxyl of substrate with solvent water. The mechanism of action of aconitase is discussed in light of this observation. Comparison shows the ENDOR study to be in agreement with previous Mössbauer and EPR spectroscopic results.

Evidence for N coordination to Fe in the [2Fe-2S] clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas.

Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties. Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical [2Fe-2S] clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J. A., Findling, K. L., Yoshida, T., Hille, R., Tarr, G. E., Hearshen, D. O., Dunham, W. R., Day, E. P., Kent, T. A., and Munck, E. (1984) J. Biol. Chem. 259, 124-133). We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins. The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz. The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz. The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings.

The identification of histidine ligands to cytochrome a in cytochrome c oxidase.

A histidine auxotroph of Saccharomyces cerevisiae has been used to metabolically incorporate [1,3-15N2] histidine into yeast cytochrome c oxidase. Electron nuclear double resonance (ENDOR) spectroscopy of cytochrome a in the [15N]histidine-substituted enzyme reveals an ENDOR signal which can be assigned to hyperfine coupling of a histidine 15N with the low-spin heme, thereby unambiguously identifying histidine as an axial ligand to this cytochrome. Comparison of this result with similar ENDOR data obtained on two 15N-substituted bisimidazole model compounds, metmyoglobin-[15N]imidazole and bis[15N]imidazole tetraphenyl porphyrin, provides strong evidence for bisimidazole coordination in cytochrome a.

Structure of cupric bleomycin. Nitrogen and proton couplings from EPR and electron nuclear double resonance spectroscopy.

Low frequency (S-band) EPR and electron nuclear double resonance (ENDOR) data have been used to provide a detailed description of the magnetic environment of the cupric complex of the antitumor antibiotic, bleomycin. S-band EPR spectra show well resolved nitrogen hyperfine structure in both gl and g regions. Computer simulation of these spectra are consistent with binding to four donor nitrogen atoms, three with AN = 10 gauss and one with AN = 15 G. ENDOR data confirm the presence of at least two inequivalent nitrogens with AN approximately 11 and 15 G. In addition, at least six proton couplings were evident in the ENDOR spectrum although these could not be unambiguously assigned. Diminution of the matrix ENDOR signal in D2O relative to that in H2O was found; this demonstrates the accessibility of H2O to the metal site.

Electron-nuclear double resonance (endor) of pitches

A preliminary study of Electron-Nuclear Double Resonance (ENDOR) has been carried out on carbonaceous pitches and their solutions. Large matrix and hyperfine ENDOR signals have been observed, the former being very sensitive to temperature, i.e. molecular motion. Measurements made over the temperature range − 100° to + 300°C indicate correlations of ENDOR response with glass transition temperatures of the pitches. The ENDOR of various concentrations of petroleum pitch in toluene suggests the presence of molecular association. Several previously unobserved spin population and phase phenomena have also been noted.

Endor study of a-Si:H

Electron-nuclear double resonance (ENDOR) experiment has been employed on undoped GD a-Si:H films before and after isochronal annealings. ENDOR signals are characterized by two different types of spectra depending on annealing temperature. It has been demonstrated through the analyses of ENDOR as well as ESR and NMR that the dangling bonds are coupled mainly with hydrogens in the broad component of NMR for high spin-density range, while those with the narrow component for low spin-density range.

Electron nuclear double resonance and electron paramagnetic resonance study on the structure of the NO-ligated heme alpha 3 in cytochrome c oxidase.

The techniques of EPR and electron nuclear double resonance (ENDOR) were used to probe structure and electronic distribution at the nitric oxide (NO)-ligated heme alpha 3 in the nitrosylferrocytochrome alpha 3 moiety of fully reduced cytochrome c oxidase. Hyperfine and quadrupole couplings to NO (in both 15NO and 14NO forms), to histidine nitrogens, and to protons near the heme site were obtained. Parallel studies were also performed on NO-ligated myoglobin and model NO-heme-imidazole systems. The major findings and interpretations on nitrosylferrocytochrome alpha 3 were: 1) compared to other NO-heme-imidazole systems, the nitrosylferrocytochrome alpha3 gave better resolution of EPR and ENDOR signals; 2) at the maximal g value (gx = 2.09), particularly well resolved NO nitrogen hyperfine and quadrupole couplings and mesoproton hyperfine couplings were seen. These hyperfine and quadrupole couplings gave information on the electronic distribution on the NO, on the orientation of the g tensor with respect to the heme, and possibly on the orientation of the FeNO plane; 3) a combination of experimental EPR-ENDOR results and EPR spectral simulations evidenced a rotation of the NO hyperfine tensor with respect to the electronic g tensor; this implied a bent Fe-NO bond; 4) ENDOR showed a unique proton not seen in the other NO heme systems studied. The magnitude of this proton's hyperfine coupling was consistent with this proton being part of a nearby protein side chain that perturbs an axial ligand like NO or O2.

EPR and electron-nuclear double-resonance studies of anF+center in sodium, potassium, and lithiumβ--alumina

EPR and electron-nuclear double-resonance (ENDOR) data from F/sup +/ centers in the conduction planes of sodium, potassium and lithium ..beta..-alumina are presented. The low-temperature Ku-band ENDOR spectra from these fast ionic conductors consist of sharp resonances from distant nuclei and broad, unresolved signals from nearest-neighbor aluminum and nearby Na, K, or Li cations in the conduction plane. The Na data are consistent with x-band ENDOR results of others. The K and Li data reveal the existence of a second inequivalent F/sup +/ center in these materials. The shape and breadth of the Na ENDOR signal can be simulated assuming that: (1) both Beevers-Ross and mid-oxygen sites are occupied, (2) the F/sup +/-ligand hyperfine interaction varies with distance as 1/R/sup 3/, and (3) there exists a Gaussian distribution of hyperfine couplings which is consistent with a 0.3-A tunneling distance inferred for Na ions from dielectric susceptibility data on Na ..beta..-alumina.

Electron nuclear double resonance signals from three ingredients of coal

ELECTRON spin resonance (ESR) spectra from coals were first reported in 19541–3. Since then many ESR studies on coals have been conducted, and the signals have been shown to arise from free radicals4,5. Recently Kevan et al. studied coals using the electron nuclear double resonance (ENDOR) technique6. They detected a single-peak signal at the free proton frequency, thus providing the first direct evidence that the environments of radicals in coals contain hydrogen atoms. We report here on ENDOR signals from three ingredients which were separated out from a coal sample. The ENDOR signal from each ingredient is substantially different from that of the other ingredients. In addition, a proton hyperfine structure (hfs) was experimentally resolved, probably for the first time, for a radical in one ingredient of a coal sample.

Nitrogen electron nuclear double resonance and proton triple resonance experiments on the bacteriochlorophyll cation in solution.

Electron nuclear double resonance signals of the pyrrole nitrogens of bacteriochlorophyll alpha cation in a solution of CH2Cl2/CH3OH (6:1) have been observed. The nitrogens are inequivalent: the hyperfine coupling constants were determined to be 2.36 and 3.18 MHz (0.84 and 1.14 G, respectively). Triple electron nuclear double resonance experiments have yielded the signs and values of nine proton hyperfine coupling constants. The assignment based on these data agrees well with previous work (Borg, D.C., Forman, A. & Fajer, J. (1976) J. Amer. Chem. Soc. 98, 6889-6893); in addition, two methine protons have been identified. Spin densities calculated from the hyperfine couplings are compared to reported molecular orbital calculations.

The determination of hydrogen coordinates in lanthanum nicotinate dihydrate crystals by Nd+3-proton double resonance

The EPR (electron paramagnetic resonance) and proton ENDOR (electron nuclear double resonance) spectra of single crystals of lanthanum nicotinate dihydrate with Nd+3 dilutely substituted for La+3 have been obtained and described by appropriate spin Hamiltonian operators. The ENDOR signals of protons lying within 5 Å of the Nd+3 have been resolved and assigned to specific protons. The physical interpretation of the measured anisotropic rare earth ion–proton hyperfine interactions has been given. The spatial coordinates of the protons in the principal axis system of the electron Zeeman interaction matrix ge have been obtained with standard deviations ranging from 0.002 to 0.015 Å, with an average of 0.006 Å. The average standard deviation for the coordinates of the 4 H2O protons nearest to the Nd+3 ion, three of which form hydrogen bonds with nicotinate nitrogen atoms, is 0.004 Å. The average standard deviation for their distances from the Nd+3 is 0.003 Å. It is clear that by means of suggested improvements of the reported techniques it should be possible to measure proton coordinates to 0.001 Å. The methods developed show promise for applications to problems concerning the structure of organic molecules, in particular organo–metallic complexes and large molecules of biological interest, such as proteins, to which paramagnetic ions may be attached.

Fluorine and proton ENDOR of aromatic radicals in solution

Electron nuclear double resonance (ENDOR) experiments were performed on fluorinated benzophenone and semi-quinone anion radicals. By detecting both fluorine and proton ENDOR signals it was possible to unravel complex hyperfine patterns. The hyperfine coupling constants obtained are discussed in the frame of MO calculations of the McLachlan and INDO type. By comparing the ENDOR enhancements for the fluorine and proton nucluei information could be obtained about the dominant relaxation mechanisms. The observed increase of ENDOR linewidths in fluorinated radicals could be explained on the basis of Freed's relaxation theory. In the fluoranil anion pronounced cross-relaxation effects were observed which are discussed to some extent.

Ligand Electron-Nuclear Double Resonance ofMn2+in CdF2

Electron-nuclear double-resonance (ENDOR) measurements have been obtained for the second, third, and fourth shells of fluorine lattice nuclei in Cd${\mathrm{F}}_{2}$: ${\mathrm{Mn}}^{2+}$ with the dc magnetic field oriented along the three high-symmetry crystalline directions. ENDOR signals from the two cadmium isotopes have also been observed. Such cadmium ENDOR signals are believed to be the first observed in a Cd${\mathrm{F}}_{2}$ lattice. Hyperfine constants for the three shells of fluorine nuclei around the ${\mathrm{Mn}}^{2+}$ defect have been obtained by fitting a simplified axial Hamiltonian. Using a more general Hamiltonian, the hyperfine constants for the two isotopes of the first shell of cadmium have also been obtained.

ENDOR Studies in Molecular Crystals. II. Computer Analysis of the Polycrystalline ENDOR Spectra of Low Symmetry Materials

The quantitative simulation of the electron nuclear double resonance (ENDOR) spectra of low-symmetry, randomly oriented molecules is discussed with particular attention paid to those systems where electron cross relaxation effectively couples the spin packets of the electron paramagnetic resonance (EPR) spectrum. ENDOR transition frequencies are calculated employing the high field approximation; frequency shifts arising from g anisotropy and the SxIx and SyIy terms of the hyperfine Hamiltonian are then evaluated by perturbation theory. ENDOR signal intensities are considered to be determined by transition moments and by electron and nuclear spin relaxation processes. The ENDOR spectra of polycrystalline samples of the x-irradiated aliphatic diacids, HOOC(CH2)nCOOH where n=1 to 8, and the alicyclic diacids, (CH2)mC(COOH)2 where m=2 to 4, have been recorded at 4.2°K and theoretical spectra computed. Reasonably good agreement is observed between experimental spectra and spectra computed assuming complete coupling of the spin packets by cross relaxation and isotropic relaxation rates. These assumptions are compatible with pulsed microwave measurements of electron relaxation rates.

Comparative Electron‐Nuclear Double Resonance Study of Two Flavoproteins

The proton electron‐nuclear double resonance (ENDOR) of flavin radicals in NADPH dehydrogenase and a flavoprotein from Azotobacter vinelandii have been studied at −160 and −120°. By combination of the apoprotein of NADPH dehydrogenase with [8‐Me‐2H3]FMN, it was demonstrated that the CH3(8) group of the coenzyme gives an ENDOR signal. The ENDOR signals from CH3(8) of the two proteins showed different hyperfine couplings which are consistent with the assignments of a radical anion, or a neutral radical enolized at 0(4), in NADPH dehydrogenase and a neutral radical protonated at N(5) in the Azotobacter flavoprotein. Analysis of the matrix‐ENDOR signals in H2O and 2H2O yields information concerning the exchangeability of protons both in the constituent water and on the apoprotein in the vicinity of the radical.

Electron-nuclear double resonance from flavin radicals in liquid and polycrystalline phase and conjugated to protein

Electron-nuclear double resonance (ENDOR) has been observed from flavin (isoalloxazine) anionic radicals and radical chelates (Zn 2+ and Cd 2+) in N, N-dimethyl-formamide solution. Hyperfine couplings to strongly coupled methyl protons could be determined and assigned by the aid of isotopically and chemically substituted compounds. No signals from other protons could be detected in the liquid phase. The methyl couplings of the anionic radical and the radical ligand of the chelates are not identical. No difference between the methyl couplings of the zinc and cadmium chelates was found. Large frozen samples showed powder ENDOR signals from rotating methyl groups. Weakly coupled protons were observed in the vicinity of the matrix ENDOR signal from the dimethylformamide protons. The low-temperature technique was extended to a flavoprotein, NADPH dehydrogenase (EC 1.6.99.1). The spectrum of prosthetic FMN radicals was similar to that from anionic radicals in polycrystalline dimethylformamide. The strongly coupled methyl group is rotating rapidly at — 160°. Indication of a preferential orientation of the ribityl chain was found. A strong matrix ENDOR signal was observed, which decreased by about 40% when 2H 2O was used as solvent. This figure should be correlated to the percent of exchangeable protons in the immediate environment of the active site. This finding together with the strength and temperature behaviour of the matrix signal indicates that the active sites are inside the protein.