Pulsed EPR Techniques Research Articles

Noncovalent and Site‐Directed Spin Labeling of Nucleic Acids

Spin label parking: The rigid spin label ç (magenta), which is an analogue of cytidine, binds site-specifically to abasic sites in duplex DNAs, through hydrogen bonding to guanine (blue) and π stacking with the flanking base pairs. EPR spectroscopy shows complete binding of ç to the abasic site at low temperatures. This new spin-labeling strategy gives easy access to spin-labeled nucleic acids for structural studies by pulsed EPR techniques.

Characterization of the semiquinone radical stabilized by the cytochrome aa3-600 menaquinol oxidase of Bacillus subtilis

Cytochrome aa3-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O2 to 2H2O. Cytochrome aa3-600 is of interest because it is a very close homologue of the cytochrome bo3 ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa3-600. Cytochrome aa3-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa3-600 in comparison with the ubi-semiquinone in cytochrome bo3. Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa3-600 in comparison with ubiquinone in cytochrome bo3. In addition, the electronic structure of the semiquinone cyt aa3-600 is more shifted toward the anionic form from the neutral state in cyt bo3.

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Cytochrome aa(3)-600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O(2) to 2H(2)O. Cytochrome aa(3)-600 is of interest because it is a very close homologue of the cytochrome bo(3) ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo(3) has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa(3)-600. Cytochrome aa(3)-600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a mena-semiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa(3)-600 in comparison with the ubi-semiquinone in cytochrome bo(3). Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa(3)-600 in comparison with ubiquinone in cytochrome bo(3). In addition, the electronic structure of the semiquinone cyt aa(3)-600 is more shifted toward the anionic form from the neutral state in cyt bo(3).

Paramagnetic centers in detonation nanodiamonds studied by CW and pulse EPR

Dispersed detonation nanodiamonds have been studied by continuous-wave (CW) and pulse EPR techniques. The spectrum of bulk radicals (g = 2.0025 ± 0.0002, a Lorentz line shape with ΔHpp = 0.95 ± 0.05 mT) dominated in CW EPR and prevented to record spectra from other paramagnetic species. The pulse EPR-spectrum was the superposition of the distorted P1-center spectrum with parameters (g = 2.0025, Axx = 2.57 mT, Ayy = 3.08 mT, Azz = 4.07 mT), the H1-center spectrum (g = 2.0028) and the single line (g = 2.0025, ΔHpp = 0.40 ± 0.05 mT) from other centers which may be assigned to surface radicals. The concentration of P1-centers has been estimated by CW EPR as 2 ± 1 ppm N.

Resolving ligand hyperfine couplings of type 1 and 2 Cu(ii) in ascorbate oxidase by high field pulse EPRcorrelation spectroscopy

Ascorbate oxidase contains two paramagnetic Cu(ii) binding sites, type 1 (T1) and type 2 (T2) and in both sites the Cu(ii) is coordinated to histidine residues. We use several pulse EPR techniques at high field (95 GHz) to determine ligand (1)H and (14)N hyperfine couplings in the two sites and identify the T1 signals by a new triple resonance correlation technique named THYCOS.

Investigating the Structural and Dynamic Properties of Membrane Proteins with Solid-State NMR, CW-EPR, ESEEM, and DEER Spectroscopic Techniques

Currently, we have very limited structural and dynamic information on membrane proteins and peptides. New spectroscopic methods are needed to probe these systems in a lipid bilayer. In order to address these issues, the Lorigan lab developing unique hybrid solid-state NMR and spin-label EPR spectroscopic techniques. Magnetic resonance spectroscopic data of 15N-, g2H-labeled and/or spin-labeled membrane proteins incorporated into vesicles and bicelles will be presented. State-of-the-art solid-state NMR and pulsed EPR techniques such as Magic Angle Spinning (MAS) NMR, Electron Spin Echo Envelope Modulation (ESEEM) spectroscopy, and Double Electron-Electron Resonance (DEER) spectroscopy will be used. The ESEEM technique can determine short to medium range distances (out to about 8 Å) between a site-specific nitroxide spin label (MTSL) and a nearby NMR-active isotopic labeled residue for a variety of different peptides and proteins which ultimately can be used to determine the difference between an α-helical and β-sheet secondary structure. DEER can be used to measure distances between 2 spin labels out to about 70 Å. The advantages and disadvantages of applying solid-state NMR and EPR spectroscopy to probe the structural and dynamic properties of membrane proteins will be discussed.

Direct Evidence for Nitrogen Ligation to the High Stability Semiquinone Intermediate in Escherichia coli Nitrate Reductase A

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR.

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfate-solubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.

Interaction and Reaction of Ethylene and Oxygen on Six-Atom Silver Clusters Supported on LTA Zeolite

Mixed silver−sodium containing zeolite LTA was prepared by aqueous ion exchange. Reduction of oxidized Ag/NaA (12 wt %) using hydrogen leads to the formation of a paramagnetic cluster possessing a well-defined structure of six equivalent Ag nuclei. The hyperfine structure of the silver clusters appeared only in Ag+-exchanged zeolite. Continuous wave and pulse EPR techniques revealed that the unpaired spin density distribution was strongly affected by adsorption of C2H4 at room temperature, and the initially symmetric structure of the reduced Ag6+ cluster turned less symmetric or less compact, lifting the equivalence of the silver atoms. The extremely weak isotropic hyperfine coupling of the protons was resolved by Mims 1H-ENDOR and HYSCORE. The temperature dependence of the electron spin−lattice relaxation was studied by pulse EPR applying a two-pulse spin−echo method. The inversion recovery of the C2H4/Ag6+ system was on the order of milliseconds, exhibiting a biexponential character. There was no effect on the hyperfine coupling of the Ag6+ cluster when it was exposed to 16O2 at room temperature. The interaction of 17O2 with the surface of the Ag6+ cluster was monitored using CW EPR. This revealed some anisotropy of the hyperfine structure of the cluster at 3.0 K. The sudden disappearance of the anisotropy in the range 7−10.5 K is ascribed to motional effects by 17O2. The hyperfine structure of the Ag6+ cluster disappeared after coadsorbing C2H4 and 16O2, and a new spectrum appeared, suggesting that an intermediate or product of a catalytic reaction is observed. In-situ EXAFS measurements were performed at the Ag K-edge of the hydrogen reduced silver clusters and after adsorbing C2H4 and coadsorbing C2H4 and O2 on the reduced clusters at room temperature. The local structural parameters were derived from a three-shell fitting model including a split Ag−Ag shell and a single Ag−O shell. Formation of a quite monodisperse structure was favored by in-situ reduction, and the structure of the reduced cluster was compatible with six silver atoms, based on the Ag−Ag coordination of four. The interatomic distance was contracted by <10% as compared to the bulk distance. The multiple-shell fitting proved the increase of the carbon plus oxygen coordination number after coadsorbing C2H4 and O2 which accounts for about one added oxygen molecule per cluster. EPR methods prove the electronic structure alterations of the clusters, seen as shifts of the confined unpaired electron spin density.

Probing Flexibility in Porphyrin-Based Molecular Wires Using Double Electron Electron Resonance

A series of butadiyne-linked zinc porphyrin oligomers, with one, two, three, and four porphyrin units and lengths of up to 75 A, have been spin-labeled at both ends with stable nitroxide TEMPO radicals. The pulsed EPR technique of double electron electron resonance (DEER) was used to probe the distribution of intramolecular end-to-end distances, under a range of conditions. DEER measurements were carried out at 50 K in two types of dilute solution glasses: deutero-toluene (with 10% deutero-pyridine) and deutero-o-terphenyl (with 5% 4-benzyl pyridine). The complexes of the porphyrin oligomers with monodentate ligands (pyridine or 4-benzyl pyridine) principally adopt linear conformations. Nonlinear conformations are less populated in the lower glass-transition temperature solvent. When the oligomers bind star-shaped multidentate ligands, they are forced to bend into nonlinear geometries, and the experimental end-to-end distances for these complexes match those from molecular mechanics calculations. Our results show that porphyrin-based molecular wires are shape-persistent, and yet that their shapes can deformed by binding to multivalent ligands. Self-assembled ladder-shaped 2:2 complexes were also investigated to illustrate the scope of DEER measurements for providing structural information on synthetic noncovalent nanostructures.

Identification of the Sites of Chlorophyll Triplet Quenching in Relation to the Structure of LHC-II from Higher Plants. Evidence from EPR Spectroscopy

The Chlorophyll a (Chl a) molecules involved in the triplet-triplet energy transfer to the central luteins in trimeric LHC-II are identified by time-resolved and pulse EPR techniques. The concept of spin angular momentum conservation during triplet-triplet energy transfer is exploited for the calculation of the spin polarization of the carotenoid triplet states. The sites with the highest probability of forming triplet states, which are quenched by the central luteins, result to be Chl603 and Chl612. "Unquenched" Chl triplet states are produced by photoexcitation in the LHC-II complex. Putative sites of these triplet states are Chl614, Chl611, Chl604, and Chl613 since they do not contribute to the formation of the observed carotenoid triplet states.

Spin labeling EPR

Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26.

Electronic Structure of the Quinone Radical Anion A1•−of Photosystem I Investigated by Advanced Pulse EPR and ENDOR Techniques

Vitamin K1 (VK1) is an important cofactor of the electron-transfer chain in photosystem I (PS I), referred to as A1. The special properties of this quinone result from its unique interaction(s) with the protein surrounding. In particular, a single H-bond to neutral A1 was identified previously in the X-ray crystal structure of PS I. During light-induced electron transfer in PS I, A1 is transiently reduced to the radical anion A1*-. In this work, we characterized the electron spin density distribution of A1*- with the aim of understanding the influence of the protein surrounding on it. We studied the light-induced spin-polarized radical pair P700*+A1*- and the photoaccumulated radical anion A1*-, using advanced pulse EPR, ENDOR, and TRIPLE techniques at Q-band (34 GHz). Exchange with fully deuterated quinone in the A1 binding site allowed differentiation between proton hyperfine couplings from the quinone and from the protein surrounding. In addition, DFT calculations on a model of the A1 site were performed and provided proton hyperfine couplings that were in close agreement with the ones determined experimentally. This combined approach allowed the assignment of proton hyperfine coupling tensors to molecular positions, thereby yielding a picture of the spin density distribution in A1*-. Comparison with VK1*- in organic solvents (Epel et al. J. Phys. Chem. B 2006, 110, 11549.) leads to the conclusion that the single H-bond present in both the radical pair P700*+A1*- and the photoaccumulated radical anion A1*- is, indeed, the crucial factor that governs the electronic structure of A1*-.

Interaction of Novel Metal Complexes with DNA: Synthetic and Structural Aspects

Metal ions bind to nucleic acids at various positions. This binding can be modulated by using metal complexes with appropriate ligands. Novel mono- and especially dinuclear metal complexes could be a powerful tool to detect rare, but still physiologically relevant, forms of DNA, e.g. the left-handed Z-DNA. In this review, our recent research activities in this area of bioinorganic chemistry are summarized. A special emphasis is laid on the synthetic challenges that arose upon the synthesis of the polyamine ligands. Further, some rather unusual approaches to elucidate the solution structure of copper bound to guanosine monophosphate with the help of pulsed EPR techniques like ENDOR and HYSCORE are described.

Multifrequency Pulsed EPR investigation of Fe-histidine Interaction of the Uniquely Coordinated [2Fe-2S] Cluster in the Outer Mitochondrial Membrane Protein, MitoNEET

Complimentary pulsed EPR techniques (ESEEM, ENDOR, HYSCORE), over multiple frequencies (X, Ka, Q-bands), were used to characterize bonding interactions of the [2Fe-2S] redox active center of the Outer Mitochondrial Membrane protein, MitoNEET. MitoNEET is the first example of a 3Cys-1His coordinated [2Fe-2S] cluster containing protein. Specifically targeting the uniquely single Fe-histidine interaction, EPR investigations integrated both natural abundance 14N and isotopically labeled 15N protein to determine the hyperfine tensor of a strongly coupled imidazole nitrogen of the bound histidine ligand. 1D-ESEEM experiments in the 31, 35GHz frequency region resulted in deep modulation patterns indicative of being near the “exact cancellation” limit and was favorable for a more direct spectral assignment of nuclear quadrupolar transition frequencies. Assignment of His87 as the bound ligand was supported by parallel experiments using H87C mutant. An additional advantage of these higher field experiments allows for greater resolved g-anisotropy and a finer degree of orientation-selected experiments, in progress. These should provide a more accurate description of the [2Fe-2S] ligand bonding interaction important for understanding the electronic structure of this new class of redox active proteins.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Population transfer for signal enhancement in pulsed EPR experiments on half integer high spin systems

High resolution pulse EPR techniques applied to half integer high spin systems, such as Mn(2+) (S = 5/2), usually focus only on the central |-1/2--> |1/2 transition. The reason is that at high fields, where the zero field splitting is considerably smaller than the Zeeman interaction, the spectrum of this transition is intense and narrow. However, because the experiments are carried out at low temperatures, the low lying levels are heavily populated and the signal of the central transition is nevertheless diminished. This, in turn affects the sensitivity of the pulse EPR technique applied. A transfer of populations from the lower lying levels, which for Mn(2+) are the |-3/2 and |-5/2 levels, to the |-1/2 level will therefore increase the sensitivity. Here we describe such an experiment, where a rapid magnetic field sweep over the |-3/2--> |-1/2 sub-spectrum is carried out, concomitantly with a low power microwave (mw) irradiation, which results in population inversion. After this sweep any pulsed EPR sequence can be applied to the central transition that now has a population difference that deviates from the equilibrium value. The feasibility of the experiment is demonstrated at W-band (95 GHz) on Mn(2+) doped in MgO for echo-detected EPR measurements and the dependence of the signal enhancement on the rate and range of the magnetic field sweep and on the mw power is described. The results are then accounted for theoretically by considering a simple fictitious spin 1/2 system. In addition, preliminary enhanced (55)Mn pulse ENDOR electron nuclear double resonance (ENDOR) spectra are presented.

Ferromagnetic spins interaction in tetraaza- and hexaazacyclophanes

Teraaza- and hexaazacyclophanes have been synthesized via a one-step palladium catalyzed amination reaction. The chemical oxidation of cyclophanes leads to the formation of radical cations, the presence of which was manifested by the appearance of new bands in UV-Vis-NIR and IR spectra. Spins of radical cations can be magnetically coupled. The nutation pulsed-EPR technique (PEANUT experiment) enables us to determine the multiplicity of the systems for different oxidant/cyclophane ratios. Spins of cyclophanes oxidized to diradical dications interact ferromagnetically to form a pure triplet state.

Evidence from EPR that nitroxide spin labels attached to human hemoglobin alter their conformation upon freezing

Human oxy-hemoglobin was spin labelled at the two β-93 cysteine positions with three different spin labels. Continuous wave electron paramagnetic resonance (CW EPR) was used to assess the dynamics of the attached spin labels in solution at room temperature. Solutions were frozen to 50 K and the distance between the spin labels was measured using the double electron electron resonance (DEER) pulsed EPR technique. The two sets of results were compared to the crystal structure of the protein and were found to be consistent with a model where the spin labels tend to adopt a less rigid surface exposed conformation in solution, but are positioned in a protein pocket in the frozen state.

Development of nickel-doped lithium formate as potential EPR dosimeter for low dose determination

EPR dosimetry employing l-α-alanine has been in vogue during the past few years, due to its tissue equivalence and linear dose response. However, l-α-alanine dosimetry has been improved during the past years, the sensitivity of this material is still too low for clinical applications. Polycrystalline lithium formate doped with NiCl 2 was therefore examined for radiation response in the dose range of clinical interest (<5 Gy) using CW EPR and pulse EPR techniques. At equal and moderate settings of microwave power and modulation amplitude lithium formate doped with 1.6 wt% of NiCl 2 was almost four times more sensitive compared to l-α-alanine, which is the most common EPR dosimeter standard. It was shown that the nickel-doped lithium formate has an excellent radiation response with a low limit of the measurable dose, and a linear dose response in the range 1–5 Gy. The relaxation and power saturation studies showed that high microwave power can be applied during measurements to improve the sensitivity of this material as an EPR dosimeter. These results show that lithium formate doped with Ni(II) exhibits promising properties required for further development of an EPR dosimeter in the dose range typical for clinical dosimetry.

Origin of magnetic moments in carbon nanofoam

A range of carbon nanofoam samples was prepared by using a high-repetition-rate laser ablation technique under various Ar pressures. Their magnetic properties were systematically investigated by dc magnetization measurements and continuous wave (cw) as well as pulsed EPR techniques. In all samples we found very large zero-field cooled-field-cooled thermal hysteresis in the susceptibility measurements extending up to room temperature. Zero-field cooled (ZFC) susceptibility measurements also display very complex behavior with a susceptibility maximum that strongly varies in temperature from sample to sample. Low-temperature magnetization curves indicate a saturation magnetization MS ≈0.35 emu g at 2 K and can be well fitted with a classical Langevin function. MS is more than an order of magnitude larger than any possible iron impurity, proving that the observed magnetic phenomena are an intrinsic effect of the carbon nanofoam. Magnetization measurements are consistent with a spin-glass type ground state. The cusps in the ZFC susceptibility curves imply spin freezing temperatures that range from 50 K to the extremely high value of >300 K. Further EPR measurements revealed three different centers that coexist in all samples, distinguished on the basis of g -factor and relaxation time. Their possible origin and the role in the magnetic phenomena are discussed.