Pulsed EPR Measurements Research Articles

Rigid Core and Flexible Terminus: STRUCTURE OF SOLUBILIZED LIGHT-HARVESTING CHLOROPHYLL a/b COMPLEX (LHCII) MEASURED BY EPR

The structure of the major light-harvesting chlorophyll a/b complex (LHCII) was analyzed by pulsed EPR measurements and compared with the crystal structure. Site-specific spin labeling of the recombinant protein allowed the measurement of distance distributions over several intra- and intermolecular distances in monomeric and trimeric LHCII, yielding information on the protein structure and its local flexibility. A spin label rotamer library based on a molecular dynamics simulation was used to take the local mobility of spin labels into account. The core of LHCII in solution adopts a structure very similar or identical to the one seen in crystallized LHCII trimers with little motional freedom as indicated by narrow distance distributions along and between α helices. However, distances comprising the lumenal loop domain show broader distance distributions, indicating some mobility of this loop structure. Positions in the hydrophilic N-terminal domain, upstream of the first trans-membrane α helix, exhibit more and more mobility the closer they are to the N terminus. The nine amino acids at the very N terminus that have not been resolved in any of the crystal structure analyses give rise to very broad and possibly bimodal distance distributions, which may represent two families of preferred conformations.

Implications for the mechanism of sulfite oxidizing enzymes from pulsed EPR spectroscopy and DFT calculations for "difficult" nuclei.

The catalytic mechanisms of sulfite oxidizing enzymes (SOEs) have been investigated by multi-frequency pulsed EPR measurements of "difficult" magnetic nuclei (35.37Cl, 33S, 17O) associated with the Mo(v) center. Extensive DFT calculations have been used to relate the experimental magnetic resonance parameters of these nuclei to specific active site structures. This combined spectroscopic and computational approach has provided new insights concerning the structure/function relationships of the active sites of SOEs, including: (i) the exchange of oxo ligands; (ii) the nature of the blocked forms; and (iii) the role of Cl- in low pH forms.

Free Radical Formation in Novel Carotenoid Metal Ion Complexes of Astaxanthin

The carotenoid astaxanthin forms novel metal ion complexes with Ca(2+), Zn(2+), and Fe(2+). MS and NMR measurements indicate that the two oxygen atoms on the terminal cyclohexene ring of astaxanthin chelate the metal to form 1:1 complexes with Ca(2+) and Zn(2+) at low salt concentrations <0.2 mM. The stability constants of these complexes increased by a factor of 85 upon changing the solvent from acetonitrile to ethanol for Ca(2+) and by a factor of 7 for Zn(2+) as a consequence of acetonitrile being a part of the complex. Optical studies showed that at high concentrations (>0.2 mM) of salt, 2:1 metal/astaxanthin complexes were formed in ethanol. In the presence of Ca(2+) and Zn(2+), salts the lifetime of the radical cation and dication formed electrochemically decreased relative to those formed from the uncomplexed carotenoid. DFT calculations showed that the deprotonation of the radical cation at the carbon C3 position resulted in the lowest energy neutral radical, while proton loss at the C5, C9, or C13 methyl groups was less favorable. Pulsed EPR measurements were carried out on UV-produced radicals of astaxanthin supported on silica-alumina, MCM-41, or Ti-MCM-41. The pulsed EPR measurements detected the radical cation and neutral radicals formed by proton loss at 77 K from the C3, C5, C9, and C13 methyl groups and a radical anion formed by deprotonation of the neutral radical at C3. There was more than an order of magnitude increase in the concentration of radicals on Ti-MCM-41 relative to MCM-41, and the radical cation concentration exceeded that of the neutral radicals.

Simultaneous acquisition of pulse EPR orientation selective spectra

High resolution pulse EPR methods are usually applied to resolve weak magnetic electron-nuclear or electron–electron interactions that are otherwise unresolved in the EPR spectrum. Complete information regarding different magnetic interactions, namely, principal components and orientation of principal axis system with respect to the molecular frame, can be derived from orientation selective pulsed EPR measurements that are performed at different magnetic field positions within the inhomogeneously broadened EPR spectrum. These experiments are usually carried out consecutively, namely a particular field position is chosen, data are accumulated until the signal to noise ratio is satisfactory, and then the next field position is chosen and data are accumulated. Here we present a new approach for data acquisition of pulsed EPR experiments referred to as parallel acquisition. It is applicable when the spectral width is much broader than the excitation bandwidth of the applied pulse sequence and it is particularly useful for orientation selective pulse EPR experiments. In this approach several pulse EPR measurements are performed within the waiting (repetition) time between consecutive pulse sequences during which spin lattice relaxation takes place. This is achieved by rapidly changing the main magnetic field, B0, to different values within the EPR spectrum, performing the same experiment on the otherwise idle spins. This scheme represents an efficient utilization of the spectrometer and provides the same spectral information in a shorter time. This approach is demonstrated on W-band orientation selective electron-nuclear double resonance (ENDOR), electron spin echo envelope modulation (ESEEM), electron–electron double resonance (ELDOR) – detected NMR and double electron–electron resonance (DEER) measurements on frozen solutions of nitroxides. We show that a factors of 3–6 reduction in total acquisition time can be obtained, depending on the experiment applied.

Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY

Transmembrane stretch M(2C) from the bacterial K(+)-translocating protein KtrB is unusually long. In its middle part, termed M(2C2), it contains several small and polar amino acids. This region is flanked by the two alpha-helices M(2C1) and M(2C3) and may form a flexible gate at the cytoplasmic side of the membrane controlling K(+) translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M(2C2) were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K(+) ions, M(2C2) residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M(2B) residue Asp-222R1 and M(2C3) residue Val-331R1 but not with respect to M(2C1) residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K(+) ions, structural models of the open and closed conformations were developed.

Frequency Dependence of Pulsed EPR Experiments.

The frequency dependence of the signal-to-noise ratio (S/N) that is theoretically possible for pulsed EPR experiments is the same as for continuous wave experiments. To select the optimum resonance frequency or frequencies for pulsed EPR experiments it is important to consider not only S/N, but also orientation selection, depth of spin echo modulation, and intensities of forbidden transitions. Evaluation of factors involved in selecting the optimum frequency for pulsed EPR measurements of distances between spins is discussed.

Interflap Distances in HIV-1 Protease Determined by Pulsed EPR Measurements

Human immunodeficiency virus type 1 (HIV-1) protease, being responsible for the cleavage of the viral polyproteins gag and gag-pol, is an essential component in the processing of viral proteins encoded in the HIV viral genome. The present work utilizes site-directed spin labeling (SDSL) and double electron−electron resonance (DEER) spectroscopy to characterize flap conformations in HIV-1 protease in an inhibited and uninhibited form. Because HIV protease is a homodimer, incorporation of the labeling probe at K55C yields a spin label pair within the protease, suitable for distance measurements by pulsed techniques. DEER experiments were performed with and without protease inhibitors to determine distances between residues 55−55‘ within the flap region of the protease. Analysis of the EPR results demonstrates that the flap region spans a continuous, rather than segmental, range of motion in its uninhibited state. Upon addition of inhibitor, the distance between spin labels shortens by ∼3−4 Å (regardless of spin label choice), indicating a closing of the flaps. More importantly, these results characterize the range of flap opening in an uninhibited form, showing distances that span 26−48 Å, indicative of a broad range of possible conformations.

Pulsed and cw EPR studies on hydrogen atoms encaged in octasilsesquioxane molecules

AbstractWe report on the synthesis of hydrogen atoms in different Si8O12R8 cages (R = (CH2)n H with n = 0–3) by both radiation chemistry and ion implantation. The efficiencies of both mechanisms have been studied quantitatively and attempts to optimise the process have been made. Spectral properties and relaxation times were investigated using both cw and pulsed EPR measurements. The Hahn Echo decay behaviour and the dependence of T 1 and T 2 on the side groups R of the cage revealed information about the predominant relaxation mechanisms. (© 2006 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Localization of the N-terminal Domain in Light-harvesting Chlorophyll a/b Protein by EPR Measurements

The conformational distribution of the N-terminal domain of the major light-harvesting chlorophyll a/b protein (LHCIIb) has been characterized by electron-electron double resonance yielding distances between spin labels placed in various domains of the protein. Distance distributions involving residue 3 near the N terminus turned out to be bimodal, revealing that this domain, which is involved in regulatory functions such as balancing the energy flow through photosystems (PS) I and II, exists in at least two conformational states. Models of the conformational sub-ensembles were generated on the basis of experimental distance restraints from measurements on LHCIIb monomers and then checked for consistency with the experimental distance distribution between residues 3 in trimers. Only models where residue 3 is located above the core of the protein and extends into the aqueous phase on the stromal side fit the trimer data. In the other state, which consequently is populated only in monomers, the N-terminal domain extends sideways from the protein core. The two conformational states may correspond to two functional states of LHCIIb, namely trimeric LHCIIb associated with PSII in stacked thylakoid membranes and presumably monomeric LHCIIb associated with PSI in nonstacked thylakoids. The switch between these two is known to be triggered by phosphorylation of Thr-6. A similar phosphorylation-induced conformational change of the N-terminal domain has been observed by others in bovine annexin IV which, due to the conformational switch, also loses its membrane-aggregating property.

Co-conformational distribution of nanosized [2]catenanes determined by pulse EPR measurements.

The co-conformational ensembles of three differently sized [2]catenanes were studied by measuring pair correlation functions corresponding to the separation of nitroxide spin labels--one attached to each of the two macrocycles--with the double electron-electron resonance (DEER) experiment. A geometric model for the [2]catenanes was derived that approximates the macrocycles by circles and takes into account the topological constraint. Comparison of the experimental to the theoretically predicted pair correlation functions gives insight into the co-conformational distribution and the size of the macrocycles. It was found that the macrocycles of the medium- and large-sized catenanes in chloroform are close to fully expanded, while they are partially collapsed in glassy o-terphenyl. For the small-sized catenane, moderate interaction between the unsaturated sections of the macrocycles in chloroform is indicated by a slight over-representation of short label-to-label separations in the pair correlation function.

Electron spin relaxation in pseudo-Jahn-Teller low-symmetry Cu(II) complexes in diaqua(L-aspartate)Zn(II).H(2)O crystals.

Low-temperature (4-55 K) pulsed EPR measurements were performed with the magnetic field directed along the z-axis of the g-factor of the low-symmetry octahedral complex [(63)Cu(L-aspartate)(2)(H2O)2] undergoing dynamic Jahn-Teller effect in diaqua(L-aspartate)Zn(II) hydrate single crystals. Spin-lattice relaxation time T(1) and phase memory time T(M) were determined by the electron spin echo (ESE) method. The relaxation rate 1/T(1) increases strongly over 5 decades in the temperature range 4-55 K. Various processes and mechanisms of T(1)-relaxation are discussed, and it is shown that the relaxation is governed mainly by Raman relaxation processes with the Debye temperature Theta(D)=204 K, with a detectable contribution from disorder in the doped Cu(2+) ions system below 12 K. An analytical approximation of the transport integral I(8) is given in temperature range T=0.025-10Theta(D) and applied for computer fitting procedures. Since the Jahn-Teller distorted configurations differ strongly in energy (delta(12)=240 cm(-1)), there is no influence of the classical vibronic dynamics mechanism on T(1). Dephasing of the ESE (phase relaxation) is governed by instantaneous diffusion and spectral diffusion below 20 K with resulting rigid lattice value 1/T(0)(M)=1.88 MHz. Above this temperature the relaxation rate 1/T(M) increases upon heating due to two mechanisms. The first is the phonon-controlled excitation to the first excited vibronic level of energy Delta=243 cm(-1), with subsequent tunneling to the neighbor potential well. This vibronic-type dynamics also produces a temperature-dependent broadening of lines in the ESEEM spectra. The second mechanism is produced by the spin-lattice relaxation. The increase in T(M) is described in terms of the spin packets forming inhomogeneously broadened EPR lines.

Twist conformational effects on the excited triplet states of aromatic ketones studied by multifrequency TREPR and pulsed EPR spectroscopy

Employing X- and Q-band time-resolved EPR and pulsed EPR techniques, we have investigated effects of twist conformation on the electronic structures and magnetic properties of the lowest excited triplet states of alkyl phenyl ketones. The conformational variation due to steric hindrance of a bulky substituent on the α-position drastically leads to a decrease of the zerofield splitting constant of |D| and an increase of the energy gap between 3nπ* and 3ππ* states. Hydrogen bonding with H2O destabilized significantly the 3nπ* state and switched the T1 state to the 3ππ* state. t-Butyl phenyl ketone gave unusually broad EPR spectra in rigid glassy matrices. The multifrequency and pulsed EPR measurements clarified that the inhomogeneously broadening spectra were ascribable to the distribution of the dihedral angle about a twist conformation. Asymmetric distribution of the ZFS parameters was interpreted in terms of the perturbation of spin—orbit interactions to the spin sublevels.

Does the Jahn-Teller effect influence electron spin relaxation? Electron paramagnetic resonance and electron spin echo studies of the Mn2+ doped (NH4)2Mg(SO4) · 6H2O single crystal and comparison with Cu2+ data

Continuous wave EPR and pulsed EPR measurements for Mn2+ in (NH4Mg(SO4)2·6H2O are presented and compared with previously published data for Cu2+. These data are analysed together, to extract an effect from Jahn-Teller dynamics on the electron spin relaxation of the Cu2+ ion. It is possible to estimate the Debye temperature of the crystal as ΘD = 240 K both for Mn2+ and Cu2+, and to prove that the vibronic dynamics do not influence T 1 for Cu2+ up to about 100 K, and only an enhancement of the phase relaxation rate 1/T M by excitations to the higher vibronic level above 20 K is observed. The spin-lattice relaxation rate is higher for Cu2+ than for Mn2+ over the whole temperature range, indicating that the g factor phonon modulation mechanism of Cu2+ relaxation is more effective than the D tensor modulation mechanism of Mn2+ relaxation. From a comparison with other relaxation data for Jahn-Teller systems, it is concluded that an enhancement of the relaxation rate is not as strong as suggested previously and can be different depending on the shape of the adiabatic potential surface.

Radical Anions of Mono- and Bis-fulleropyrrolidines: An EPR Study

The radical anions of three C60 derivatives (N-methyl-3,4-fulleropyrrolidine, N-methyl-2-(3,6,9-trioxadecyl)3,4-fulleropyrrolidine, and bis-N-methyl-3,4-fulleropyrrolidine are respectively MFP - , tegMFP - , and bisMFP - ) have been investigated by continuous wave (cw) and pulsed EPR in methyltetrahydrofuran and tetrahydrofuran solutions. The EPR spectra for MFP - and tegMFP - show a 1:1:1 triplet, due to the hyperfine coupling with the pyrrolidine nitrogen. The EPR spectrum of bisMFP - shows a superimposition of two signals, each due to a 1:2:3:2:1 quintet. Each quintet is due to the hyperfine coupling with the two pyrrolidine nitrogen atoms. The two signals are attributed to two different conformers of the radical. A direct transfer of spin density from the C60 sphere to the nitrogen atoms is discussed as arising from the bent conformation of the pyrrolidine rings. All the spectra show several weaker satellite lines, due to the radical anions containing a 13 C atom. From their analysis a C 2V symmetry for the spin distribution is obtained in the case of MFP - and tegMFP - . The EPR line widths of these latter radicals at room temperature are larger than those expected on the basis of spin relaxation due to rotational diffusion in solution, and the line widths decrease upon decreasing the temperature. Pulsed EPR measurements of T1 in liquid and frozen solutions show that the EPR line widths are determined by the anomalously short lifetimes of the spin states. The temperature dependence of T1 obeys to the Arrhenius law, giving an activation energy of 7 kJ/mol. The latter results are compared with those obtained in the past for the lone C60 . They are attributed to the closeness of the electronic levels, due to the weak perturbation of the pyrrolidine ring on the symmetrical electronic distribution of C 60. On the other hand, the spin relaxation properties of bisMFP - are shown those expected for a nonsymmetrical radical. The comparison of cw- and pulsed-EPR spectra of MFP - in frozen solutions of MeTHF and THF indicates the presence of aggregates of interacting radicals in THF.

Transient and pulsed EPR spectroscopy on the radical pair state P 865 +. Q A −. to study light-induced changes in bacterial reaction centers

The radical pair state P 865 +. Q A −. (P865: primary donor, QA: quinone acceptor) in Zn-substituted bacterial reaction centers is investigated using transient and pulsed EPR spectroscopy. For photoexcited samples not frozen in the dark but under continuous illumination, a prolonged lifetime of this radical pair state is observed in agreement with previous studies using time resolved optical spectroscopy. The transient EPR spectra revealed neither a different orientation of the quinone acceptor anion nor a change of itsg-anisotropy in the sample frozen in the charge separated state as compared with that frozen in the dark. The latter finding indicates a similar hydrogen bonding situation for Q A −. in both samples. Changes observed in the transient EPR spectra are interpreted as result of contributions from spin-polarized Q A −. which was generated in part of the sample while freezing under illumination. From the out-of-phase echo modulation pattern observed in the pulsed EPR measurements, it follows that the distance between P 865 +. and Q A −. is the same in dark frozen samples and in those frozen under continuous illuminaton. This is in contrast to the model suggested by Kleinfeld D., Okamura M.Y., Feher G.: Biochemistry23, 5780 (1984), in which an increased distance and a larger distribution of distances was suggested for samples frozen under illumination. The prolonged lifetime of the radical pair P 865 +. Q A −. is discussed in terms of differences in the relaxation behaviour of the protein.

Pulsed EPR study of the S′ 3 signal in the Ca 2+-depleted photosystem II

The Ca 2+-depleted photosystem II (PS II) was studied by pulsed EPR spectroscopy. A short (5 s) illumination of the PS II in the S′ 2 state resulted in formation of the doublet S′ 3 signal at g ≈ 2.02 with a splitting of about 15 mT concomitant with the decrease of the modified Mn multiline signal by about 40%. Further illumination up to three minutes led to the formation of another S′ 3 signal with a singlet-like feature at g ≈ 1.98 and to the complete disappearance of the modified Mn multiline signal. Computer simulations of the shape of the doublet S′ 3 signal and the dependence of its electron spin echo amplitude on the microwave field strength suggest that dipole and exchange interactions between two organic radicals are responsible for the doublet S′ 3 signal. The pulsed ENDOR-induced EPR measurements indicate that the ENDOR spectrum of the tyrosine radical Y Z + is associated with the doublet S′ 3 signal only.

Evidence of the Jahn-Teller splitting of C60- in C60-tetraphenylphosphoniumchloride from an electron-spin-relaxation study.

Pulsed EPR measurements of the transverse and longitudinal relaxation times of the ${\mathrm{C}}_{60}^{\mathrm{\ensuremath{-}}}$-anion radical in crystalline ${\mathrm{C}}_{60}$-tetraphenylphosphoniumchloride were done at temperatures from 4 to 40 K. Above 40 K to room temperature the longitudinal relaxation time was taken from the cw-EPR linewidth. The low-temperature data are explained in terms of local magnetic fluctuations, slow ${\mathrm{C}}_{60}^{\mathrm{\ensuremath{-}}}$ motion, and localized two-level states related with the local disorder. The relaxation data at higher temperatures reveal experimental evidence for the Jahn-Teller distortion of the ${\mathrm{C}}_{60}^{\mathrm{\ensuremath{-}}}$-anion radical and allow to determine the Jahn-Teller splitting between the ${\mathit{a}}_{2\mathit{u}}$ electronic ground state and the excited ${\mathit{e}}_{1\mathit{u}}$ state of the unpaired electron.

Interaction of spin labels with transition metals: Part 2

Interaction of spin labels with transition metals: Part 2