The g-matrix of photosynthetic pigments has been studied in the last decade due to the advent of high-field EPR techniques. It can be observed when the spectral splitting of the principal g-factor components is larger than the linewidth due to unresolved hyperfine splitting and if there is no g-strain obscuring it. For large organic molecules such as the primary electron donor in photosynthetic reaction centers (RC) this usually requires fields above 11 T, or, for fields between 3 and 11 T, full deuteration and/or single crystal work. Here we present for the first time the fully resolved rhombic EPR spectrum of the primary donor of Blastochloris viridis (formerly called Rhodopseudomonas viridis), a purple photosynthetic bacterium containing bacteriochlorophyll b. As was the case for Rhodobacter sphaeroides, g-strain is negligible for this radical up to a field of 24 T. The temperature dependence of the g-anisotropy is presented and compared with that of the bacteriochlorophyll a-containing Rb. sphaeroides and plant photosystem I. A slight shift in the principal components of the g-matrix is observed at temperatures below 70 K, where it becomes more axial. The experimental work is complemented with theoretical calculations for g using the semi-empirical INDO/S method as implemented in the program ZINDO. The theoretical results generally agree well with the experiment. This indicates that a satisfactory description of the anisotropic g-tensor for radical cations of large planar molecules like the chlorophylls as well as their aggregates, e.g., reaction center primary donor special pairs, is possible with this relatively cheap semi-empirical approach.

High-field EPR experiments at 12 T/330 GHz were performed on chlorophyll a radical cations in methylene chloride at low temperatures. Using fully deuterated chlorophyll it was possible to obtain the principal components of the rhombic g-tensor as gα = 2.00329 ± (5 × 10-5), gβ = 2.00275 ± (8 × 10-5), and gγ = 2.00220 ± (8 × 10-5). Protonated chlorophyll radicals did not give enough spectral resolution to yield the g-anisotropy from the EPR spectrum. This was true even at higher field/frequency combinations up to 24 T/670 GHz. Semiempirical calculations were performed using the INDO/S method (ZINDO) which yielded good agreement with the experimental data.

High-field EPR experiments at 12 T/330 GHz were performed on chlorophyll a radical cations in methylene chloride at low temperatures. Using fully deuterated chlorophyll it was possible to obtain the principal components of the rhombic g-tensor as g(alpha) = 2.00329 +/- (5 x 10(-5)), g(beta) = 2.00275 +/- (8 x 10(-5)), and g(gamma) = 2.00220 +/- (8 x 10(-5)). Protonated chlorophyll radicals did not give enough spectral resolution to yield the g-anisotropy from the EPR spectrum. This was true even at higher field/frequency combinations up to 24 T/670 GHz, Semiempirical calculations were performed using the INDO/S method (ZINDO) which yielded good agreement with the experimental data.

The g-matrix of a free radical is an important observable that yields information on its electronic structure. It is usually measured by electron paramagnetic resonance (EPR) under "high field" conditions, where the spectral splitting of the principal g-factor components is larger than the line width due to unresolved hyperfine splitting. For large organic molecules such as the primary electron donor in photosynthetic reaction centers (RC) this usually requires fields above ii T, or, for fields between 3 and 11 T, full deuteration and/or single-crystal work. When trying to obtain improved spectral resolution a major concern is the presence of g-strain which leads to extra line broadening. Here we show that g-strain is negligible for bacterial RCs up to a field of 24 T. We investigated the temperature dependence of the g-anisotropy for RCs from Rhodobacter (Rb.) sphaeroides using different detergents and find that within experimental errors there is no change in the principal g-matrix components up to 200 K. This is the first report of a successful EPR experiment on a biological sample above the limits of superconducting magnets.

The 95-670 GHz EPR measurements at 5 K were performed on canthaxanthin radical cation chemically generated on silica-alumina. The 327 GHz and higher frequency EPR spectra were resolved into two principal components of the g tensor. Spectral simulation indicated this to be the result of 8 anisotropy where g(parallel to) 2.0032 and g(perpendicular to) = 2.0023. This type of g tensor is consistent with the theory for polyacene pi-radical cations, which states that the g tensor becomes cylindrically symmetric with increasing chain length. This also demonstrates that the symmetrical unresolved EPR line at 9 GHz is due to a carotenoid pi-radical cation with electron density distributed throughout the whole chain as predicted by RHF-INDO/SP molecular orbital calculations. The lack of temperature dependence of the EPR line widths over the range of 5-80 K at 327 GHz suggests rapid rotation of methyl groups even at 5 K that averages out the proton couplings from three oriented beta-protons. In fact, similar line widths at 5 K were observed at 670 GHz. Simulation of EPR spectra at 95-250 GHz gives only symmetrical unresolved lines. The present work shows that the 327-670 GHz EPR measurements are sufficient to resolve the individual g tensors of C-H containing pi-radicals in powder and frozen glasses. Symmetry differences can be deduced from which radical identification can be made.

Structural and biochemical characterization of the nonliganding residue glutamine 143 near the manganese of human Mn superoxide dismutase (hMnSOD), a homotetramer of 22 kDa, reveals a functional role for this residue. In the wild-type protein, the side-chain amide group of Gln 143 is about 5 A from the metal and is hydrogen-bonded to Tyr 34, which is a second prominent side chain adjacent to the metal. We have prepared the site-specific mutant of hMnSOD with the conservative replacement of Gln 143 --> Asn (Q143N). The crystal structure of Q143N shows that the side-chain amide nitrogen of residue 143 is 1.7 A more distant from the manganese than in the wild-type enzyme. The Tyr 34 side-chain hydroxyl in Q143N is also moved to become 0.6 A more distant from the metal due to an additional water molecule. Differential scanning calorimetry showed that Q143N is slightly more stable than the wild-type enzyme with Tm for the main unfolding transition increased by 2 degrees C to 90.7 degrees C. Pulse radiolysis and stopped-flow spectrophotometry reveal that unlike wild-type hMnSOD, which is strongly inhibited by peroxide, Q143N MnSOD exhibits no product inhibition even at concentrations of O2. - in the millimolar range, and its catalysis follows Michaelis kinetics with no evidence of cooperativity. However, the overall catalytic activity of this mutant was decreased 2-3 orders of magnitude compared with the wild-type MnSOD, which can account for its lack of product inhibition. Q143N MnSOD lacked the visible absorption spectrum typical of wild-type Mn(III)SOD. Also, unlike the wild-type Mn(III)SOD, which is electron paramagnetic resonance (EPR) silent, Q143N MnSOD has a complex EPR spectrum with many resonances in the region below 2250 G. We conclude that the Gln 143 --> Asn mutation has increased the reduction potential of manganese to stabilize Mn(II), indicating that Gln 143 has a substantial role in maintaining a reduction potential favorable for the oxidation and reduction cycles in the catalytic disproportionation of superoxide. A solvent hydrogen isotope effect near 2 for kcat in catalysis by Q143N hMnSOD indicates rate-contributing proton transfers to form product hydroperoxide anion or hydrogen peroxide. The data demonstrate a prominent role for Gln 143 in maintaining the microenvironment of the manganese and in efficient catalysis of superoxide dismutation to oxygen and hydrogen peroxide.

Pulsed EPR spectroscopy was used to investigate the relaxation properties of the electron transfer components of photosystem 1. The magnetic dipolar interactions between the quinone, A1, and the redox active chlorophylls, P700 and A0, and the iron-sulphur component Fe-SX, were investigated. The spin lattice relaxation transients for A0−⋅, A1−⋅, and P700+⋅ were non-single-exponential in the presence of reduced iron-sulphur centres, suggesting a dipolar magnetic interaction. However, when the iron-sulphur centres were removed the relaxation transients for these species became mono-exponential demonstrating that the iron sulphur centres were the relaxation enhancing species. When Fe-SX was reduced it became the dominant relaxation enhancing species. Qualitatively the relaxation enhancement is similar for A0−⋅ and A1−⋅ with the least effect on P700+⋅. Quantitative analysis based on the procedure described by Hirsh et al. (Biochemistry 31 [1992] 532–541), provides estimates of the P700 − Fe-SX distance of 25–35 Å, A1 − Fe-SX and A0 − Fe-SX of 20–30 Å. The relevance of these distances to those determined by other spectroscopic techniques and X-ray crystallography is discussed.

It has recently been shown that some galactose-specific plant lectins protect thylakoid membranes from freeze–thaw damage [D. K. Hinchaet al. Plant Physiol.103,59–65 (1993)]. In the present investigation, theRicinus communisseed lectins RCA60and RCA120have been used to investigate further the effects of lectin binding on the physical properties of thylakoids. Both proteins bind to digalactolipids, but only RCA60is cryoprotective. We found that RCA60reduced the glucose permeability of the membranes. Fluorescence depolarization measurements with the probe trimethylammonium-diphenylhexatriene (TMA-DPH) showed a significant reduction of lipid fluidity in the presence of RCA60, but not with RCA120. The fluidity of the hydrocarbon core region of thylakoids, as probed with DPH, was not influenced. These results were corroborated by electron spin resonance spectroscopy using doxyl stearic acids (DSA) with the reporter group located in position 5, 12, or 16 of the fatty acyl chains. Clear effects of RCA60were only apparent with 5DSA. When we used the fluorescence probe merocyanine 540 to investigate the lipid packing density in the glycerol backbone region of the membrane, we found a decrease in fluorescence emission in the presence of RCA60. We conclude from these data that cryoprotective lectins lead to changes in the packing of the membrane lipids in the interfacial region which result in a lower solute permeability of the membranes.

In this present study we report millimeter and submillimeter high-field CW EPR spectra of P700(.+), the primary electron donor in photosystem I. The data shown are the first well-resolved chlorophyll radical spectra observed without prior deuteration. The signal was generated from isolated plant photosystem I (in both digitonin and Triton-X100 preparations) by photooxidation. At 325 GHz and higher frequencies, the spectrum was resolvable into the three principal components of its g-matrix. They were obtained to high accuracy by spectral simulation and were found to be g(xx) = 2.003 17(+/-7 x 10(-5)), g(yy) = 2.002 64(+/-7 x 10(-5)), g(zz) = 2.002 26(+/-7 x 10(-5)) at 40 K and g(xx) 2.003 07(+/-7 x 10(-5)), g(yy) = 2.002 60(+/-7 x 10(-5)), g(zz) = 2.002 26(+/-7 x 10(-5)) at 200 K. These values indicate a temperature dependence of the g-values in P700(+) that was measured over a temperature range of 5-260 K using both preparations. The results showed that g(xx) changed with temperature while g(yy) and g(zz) remain constant within the error margin, suggesting that the P700(.+) radical becomes less anisotropic with increasing temperature.

The association of water with the Mn of the water oxidizing complex was investigated using H2(17)O- and 2H2O-reconstituted lyophilized photosystem II particles. The pulsed electron paramagnetic resonance (EPR) technique of electron spin echo envelope modulation (ESEEM) was used to investigate the interaction of the magnetic 2H and 17O nuclei with the paramagnetic S2 state of the Mn complex and other photosystem II components. ESEEM offers a much more specific and sensitive detection of this type of interaction than continuous wave (CW) EPR. Unlike earlier reports using CW EPR, these experiments did not detect any interaction of water with the multiline EPR signal from the S2 state of the Mn complex. No signals indicating specific interaction of either H or O with the multiline signal were detected. Signals due to 2H and 17O were detected only at the Larmour frequency, indicating nonspecific "distant ENDOR" effects. A weak interaction with 17O was detected both in S1, when the Mn is EPR silent, and in S2, but only on the high-field side of g = 2. This interaction may be with the Rieske iron-sulfur center in the cytochrome b6f complex. The results were the same whether the multiline signal was generated by 200 K illumination of dark-frozen samples, or by room temperature illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Illumination at room temperature in the presence of an electron acceptor to allow multiple turnovers of the system with cycling of the S states did not result in the appearance of any new interactions. These results appear to exclude close (less than 6 A) binding of water to the Mn center giving rise to the multiline signal, and also to exclude mechanisms in which water oxidation involves the breaking and re-formation of the mu-oxo bridges of the Mn complex. They cannot, however, exclude models in which water binding to the manganese complex and direct oxidation by the manganese complex occur in the higher S states, or are catalyzed by one bis(mu-oxo) Mn dimer while oxidizing equivalents are accumulated in the S2 state by a second bis(mu-oxo) Mn dimer.

The electronic structure of the oxidized primary chlorophyll electron donor, P840 +. , of the green sulfur bacterium Chlorobium limicola has been investigated using electron spin echo envelope modulation (ESEEM) spectroscopy. This ESEEM investigation of the electron spin density distribution in the radical cation P840 +. in membranes isolated from C. limicola confirms that the electron spin is shared eqully between the two bacteriochlorophyll a molecules. Observation of the small hyperfine couplings to the ring nitrogens by ESEEM gives results that are in agreement with those obtained from ENDOR measurements (S. E. J. Rigby, R. Thapar, M. C. W. Evans and P. Heathcote, FEBS Lett. 350,24–28, 1994) of the large hyperfine couplings to the methyl group protons. These results in combination with the Raman spectroscopy of P840 (U. Feiler, D. Albouy, B. Robert and T. A. Mattioli, Biochemistry 34,11099–11105, 1995) all indicate that the reaction center of green sulfur photosynthetic bacteria is functionally a protein homodimer providing a symmetrical protein environment for the primary electron donor.

Individual EPR properties of the four hemes in the cytochrome subunit of the Rhodopseudomonas viridis reaction centre have been studied. EPR signals of the four hemes were resolved and assigned in the isolated reaction centres: g(x) = 3.05 and g(y) = 2.20, E(m) = 394 mV; g(x) = 3.39, E(m) = 325 mV; g(x) = 3.31, E(m) = 5 mV; g(z) = 3.28, E(m) = -96 mV. It has been found that the highest potential heme may appear in two interconvertible EPR forms with g(z) = 3.05, g(y) = 2.20 and g(z) = 3.16, g(y) = 2.09; the latter dominates in glycerol-containing medium. Methionine ligand replacement by exogenous imidazole has been performed in the methionine-histidine coordinated lowest potential heme. This gives a three component EPR spectrum characteristic of bis-imidazole complexes. It is suggested that the distinctive EPR signal of the other low potential heme with two histidine ligands (and only the g, component detectable at g = 3.31) is determined by the relative orientation of the coordinating histidine imidazole ring planes which make an angle of 64 degrees.Direct spin-lattice relaxation rates measurement in the cytochrome subunit showed that the heme with E(m) = 394 mV is characterized with T-1 of 49 mu s at 9.6 K while the other three hemes relax as one component with T-1 of approximate to 15 mu s; this implies that the highest potential heme is magnetically isolated in the protein.

Manganese K-edge X-ray spectra have been obtained for Photosystem II samples depleted of calcium by various NaCl treatments which inhibit oxygen evolution without displacement of managanese. Changes in the pre-edge feature due to 1s → 3d transitions and shifts in the edge position of samples in the S∗1, S∗2 and S∗3 states indicate managanese oxidation for the S∗1 → S∗2 and S∗2 → S∗3 transitions. Analysis of the EXAFS shows changes on NaCl treatment compared to native PS II membranes which are further modified by the chelator, EGTA. The intensity of the Fourier transform peak at about 1.8 Å, assigned to oxygen, increases with increasing S-state in agreement with oxidation state changes, although the average distance for this first shell remains constant. Each of the inhibitor-treated S-states have a short average Mn-O bond length, showing the retention of the μ-oxo bridges postulated to occur in native samples. The Mn-Mn shell, found at 2.7 Å in native PS II membranes is split in NaCl-treated samples to give a 2.7 Å Mn-Mn and 3.0 Å Mn-X interaction (X = Mn,C/O/N). Splitting of the 2.7 Å shell is most apparent in the higher S-states, S∗3 >S∗2 >S∗1. Although the scatterers at 3.0 Å could not be uniquely identified, the intensity favours heavy scatterers, Mn/Ca, over light scatterers, C/O/N. The cluster appears to contain at least two inequivalent Mn-Mn pairs or shows multiple scattering from a ligand such as tyrosine/histidine. NaCl treatment results in a smaller 3.33.6 Å intensity compared to untreated PS II samples which could be due to replacement of calcium scatterers at this distance and/or a structural rearrangement. EGTA addition results in an S∗2 state with a modified EPR spectrum but has only a small effect on the XAS. The changes on removal of the 17 and 23 kDa extrinsic polypeptides are small compared to the effect of the calcium depletion/NaCl treatment, indicating a minor role for these polypeptides on the structure of the cluster. Changes in the electron spin lattice relaxation time, T1 of the dark stable tyrosine radical YD have also been studied using pulsed EPR. The T1 relaxation times decreased with increasing modified S-state S∗1 >S∗2 >S∗3, indicating oxidation occurring at or near the manganese cluster.

Conference Article| May 01 1994 Photosystem II electron transfer: the manganese complex to P680 Jonathan H. A. Nugent; Jonathan H. A. Nugent 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Peter J. Bratt; Peter J. Bratt 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Michael C. W. Evans; Michael C. W. Evans 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Dugald J. MacLachlan; Dugald J. MacLachlan 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Stephen E. J. Rigby; Stephen E. J. Rigby 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Stuart V. Ruffle; Stuart V. Ruffle 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Sandra Turconi Sandra Turconi 1Department of Biology, Darwin Building, University College London, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1994) 22 (2): 327–331. https://doi.org/10.1042/bst0220327 Article history Received: December 20 1993 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Jonathan H. A. Nugent, Peter J. Bratt, Michael C. W. Evans, Dugald J. MacLachlan, Stephen E. J. Rigby, Stuart V. Ruffle, Sandra Turconi; Photosystem II electron transfer: the manganese complex to P680. Biochem Soc Trans 1 May 1994; 22 (2): 327–331. doi: https://doi.org/10.1042/bst0220327 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: OEC, oxygen-evolving complex, Chl, chlorophyll; Yz, Tyr-161 on the D1 polypeptide, YD, Tyr-161 on the D2 polypeptide, photosystem II, PSII, e.n.d.o.r., electron nuclear double resonance This content is only available as a PDF. © 1994 Biochemical Society1994 Article PDF first page preview Close Modal You do not currently have access to this content.

Conference Article| August 01 1994 Metal—Redox Centre interactions in photosynthetic reaction centres Michael C. W. Evans; Michael C. W. Evans * *To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Matthew C. Berry; Matthew C. Berry 1Department of Biology, University College London, Gower Street, London WCIE 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Peter J. Bratt; Peter J. Bratt 1Department of Biology, University College London, Gower Street, London WCIE 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Olga Kaminskaya; Olga Kaminskaya 1Department of Biology, University College London, Gower Street, London WCIE 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Jonathan H. A. Nugent Jonathan H. A. Nugent 1Department of Biology, University College London, Gower Street, London WCIE 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1994) 22 (3): 718–720. https://doi.org/10.1042/bst0220718 Article history Received: March 21 1994 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Michael C. W. Evans, Matthew C. Berry, Peter J. Bratt, Olga Kaminskaya, Jonathan H. A. Nugent; Metal—Redox Centre interactions in photosynthetic reaction centres. Biochem Soc Trans 1 August 1994; 22 (3): 718–720. doi: https://doi.org/10.1042/bst0220718 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1994 Biochemical Society1994 Article PDF first page preview Close Modal You do not currently have access to this content.

Electron spin resonance spectra of a series of x-doxylstearic acids (x = 5, 7, 10, 12, and 16), solubilized in anionic dihexadecyl phosphate vesicles containing 10, 30, and 50 mol % cholesterol, are analyzed by employing spectral line-shape simulation. The spectra from the vesicle phase are well reproduced by using the microscopic order-macroscopic disorder model of Freed and co-workers with Brownian rotational diffusion. The partial averaging of the magnetic interactions by local anisotropic motions in the vesicles is quantified mainly by the order parameter (S) and the rotational diffusion rate perpendicular to the alkyl chain (R(perpendicular-to)). The simulations show that the order parameter decreases with increasing x, suggesting a relatively extended conformation, unlike the U-shaped, bent conformation of the x-doxylstearic acid alkyl chain found previously in cationic dioctadecyldimethylammonium chloride vesicles. Furthemore, the headgroup region of the vesicle is less ordered in anionic dihexadecyl phosphate than in cationic dioctadecyldimethylammonium chloride vesicles. This result in liquid vesicle solutions corroborates the same x-doxylstearic conformations found in frozen vesicle solutions by electron spin echo modulation spectroscopy. The effect of cholesterol is found to decrease the observed order parameter for all doxyl positions. This effect is attributed to more water penetration into the vesicle interface arising from the intercalation of the large rigid cholesterol molecule. The effect of cholesterol addition is supported by using 3-doxyl-5alpha-cholestane, a spin probe which resembles cholesterol. It is found that the addition of cholesterol up to 50 mol % has little effect on the observed electron spin resonance spectrum of cholestane.

A pulsed EPR saturation-recovery method has been used to measure spin-lattice relaxation times (T1) for aminoxyl spin labels in cardiolipin bilayers, with and without cytochrome c. The relaxation time for each spin label was determined at various positions in the bilayer, which included the bilayer surface and three positions within the hydrophobic interior in the membrane, as well as a position close to the glycerol backbone of phospholipids in the bilayer. A dynamic profile for the hydrocarbon chains in bilayers was found in agreement with results from other techniques. On addition of cytochrome c to cardiolipin bilayers, changes in the spin-lattice relaxation time and order parameter were observed around the unsaturated region in the cardiolipin acyl chains. Supported by complementary lineshape analysis, these effects were interpreted as changes in the molecular dynamics around the double bonds in the acyl chains of cardiolipin bilayers, induced upon the binding of cytochrome c.

Electron spin resonance spectra of a series of x-doxylstearic acids (x = 5, 7, 10, 12, and 16) solubilized in cationic dioctadecyldimethylammonium chloride vesicles were recorded 298-328 K and analyzed by employing spectral line simulation. The spectra from the vesicle phase are well reproduced by using the microscopic order-macroscopic disorder model of Freed and co-workers with Brownian rotational diffusion. The partial averaging of the magnetic interactions by local anisotropic motions in the vesicles is quantified mainly by an order parameter (S) and the rotational diffusion rate perpendicular to the alkyl chain (R perpendicular-to). The simulations show that the doxyl position becomes progressively less ordered (S decreases) as x increases up to x = 12 but inverse more ordered at x = 16. This suggests a U-shaped bent conformation of the doxylstearic acid alkyl chain. R perpendicular-to shows a similar, but inverse, correlation; that is, R perpendicular-to increases, plateaus, and decreases with increasing x. The U-shaped conformation of the spin probe alkyl chain in liquid vesicle solutions is the same as deduced previously in frozen vesicle solutions measured by electron spin echo modulation spectroscopy.