Electron Nuclear Double Resonance Spectroscopy Research Articles

Electron spin resonance and electron nuclear double resonance studies of flavoproteins involved in the photosynthetic electron transport in the cyanobacterium Anabaena sp. PCC 7119.

The flavins of ferredoxin-NADP+ reductase (FNR) and flavodoxin from the cyanobacterium Anabaena PCC 7119 were obtained in their semiquinone states at pH 7 by photoreduction of the pure proteins in the presence of EDTA and 5-deazariboflavin. For FNR, the ESR signal of the FAD semiquinone was centred at g = 2.005 with linewidths 2.0 mT in H2O and 1.48 mT in D2O. These data are in agreement with those reported for other neutral flavin semiquinones. The linewidths were the same when measured either at X-band (9.35 GHz) or at S-band (4 GHz), indicating that line broadening is due to unresolved nuclear hyperfine couplings, caused in part by exchangeable protons. When the substrate, NADP+, was added to the semiquinone form of the protein no changes in the linewidth or shape of the spectra were detected, but a decrease in the ESR signal due to the FNR semiquinone was observed, consistent with the reduction of NADP+ to NADPH by reduced FNR and, subsequent displacement of the equilibrium. No changes in the shape or linewidth of the FNR ESR signals were observed when photoreduction of FNR was performed in the presence of either flavodoxin or ferredoxin. Electron nuclear double resonance (ENDOR) spectroscopy of FNR semiquinone from Anabaena PCC 7119 provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 5-9.5 MHz, is attributed to protons on C6 and on 8-CH3 of the flavin ring. No change in these hyperfine couplings was detected when the protein was studied in D2O, but the coupling Aiso attributed to protons on 8-CH3 decreased from 8.12 MHz to 7.72 MHz in the presence of NADP+. The decrease in the electron spin density distribution on this part of the flavin ring system was attributed to binding of the substrate, polarising the electron density distribution of the flavin towards the pyrimidine ring. A second group of signals was observed, with hyperfine couplings less than 3 MHz, some of which disappeared when the protein was transferred into D2O. Effects of NADP+ binding to the protein were also observed in these weak couplings. These signals are attributed to displaced water protons, or to exchangeable protons from amino acid residues on the protein near the flavin-binding site, involved in substrate stabilization.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of calcium-binding sites in the kidney stone inhibitor glycoprotein nephrocalcin with vanadyl ions: electron paramagnetic resonance and electron nuclear double resonance spectroscopy.

Nephrocalcin (NC) is a calcium-binding glycoprotein of 14,000 molecular weight. It inhibits the growth of calcium oxalate monohydrate crystals in renal tubules. The NC used in this study was isolated from bovine kidney tissue and purified with the use of DEAE-cellulose chromatography into four isoforms, designated as fractions A-D. They differ primarily according to the content of phosphate and gamma-carboxy-glutamic acid. Fractions A and B are strong inhibitors of the growth of calcium oxalate monohydrate crystal, whereas fractions C and D inhibit crystal growth weakly. Fraction A, with the highest Ca(2+)-binding affinity, was characterized with respect to its metal-binding sites by using the vanadyl ion (VO2+) as a paramagnetic probe in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopic studies. By EPR spectrometric titration, it was shown that fraction A of NC bound VO2+ with a stoichiometry of metal:protein binding of 4:1. Also, the binding of VO2+ to NC was shown to be competitive with Ca2+. Only protein residues were detected by proton ENDOR as ligands, and these ligands bound with complete exclusion of solvent from the inner coordination sphere of the metal ion. This type of metal-binding environment, as derived from VO(2+)-reconstituted NC, differs significantly from the binding sites in other Ca(2+)-binding proteins.

Comparative Study of Reaction Centers from Photosynthetic Purple Bacteria: Electron Paramagnetic Resonance and Electron Nuclear Double Resonance Spectroscopy

Reaction centers (RCs) from four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and the recently discovered bacterium Rhodospirillum centenum, have been characterized by optical spectroscopy [Wang, S., Lin, X., Woodbury, N. W., & Allen, J. P. (1994) Photosynth. Res. (submitted for publication)] and magnetic resonance spectroscopy. All RCs contain a bacteriochlorophyll (BChl) a dimer as the primary donor. For Rb. sphaeroides and Rs. rubrum the donor QY optical band is at approximately 865 nm, compared to approximately 850 nm for Rb. capsulatus and Rs. centenum. The primary donor in the RCs can be converted between these two forms by the addition or removal of charged detergents. The electronic structure of the cation radical of the primary electron donor P+. was investigated in these species using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron nuclear triple resonance (TRIPLE) spectroscopy. The EPR line widths of P+. vary significantly and the ENDOR and Special TRIPLE spectra reveal drastic differences in the spin density distribution of the dimer for the different species. Reaction centers from Rb. sphaeroides and Rs. rubrum have a slightly asymmetric spin density distribution over the two halves of the dimer. The respective ratios are 2:1 and 1.6:1 in favor of the L-half of the BChl a dimer. In contrast, the spectra of P+. in reaction centers from Rb. capsulatus and Rs. centenum show an almost complete localization of the unpaired electron on the L-half of the dimer (ratio approximately 5:1).(ABSTRACT TRUNCATED AT 250 WORDS)

ENDOR and special triple resonance studies of chlorophyll cation radicals in photosystem 2.

Electron nuclear double resonance (ENDOR) and special triple (ST) resonance spectroscopies have been used to study the cation radicals of the primary donor, P680, and two secondary donor chlorophylls (Chl) in photosystem 2 (PS2). Two different preparations were employed, Tris-washed PS2 membranes and PS2 reaction centers (D1-D2-I-Cytb559 complex). One secondary donor Chl a cation radical, Chl1.+, was generated in the Tris-washed preparation, while the P680.+ radical cation and a further Chl a cation radical, Chl2.+, were produced in the reaction center preparation. The ENDOR spectrum of the primary donor radical cation of photosystem 1 (P700.+) is also presented for comparison. Hyperfine coupling constants for methyl groups have been measured for all three PS2 radical species and assigned by comparison with previously published spectra of Chl a radicals in vitro. Electron spin densities were calculated from these hyperfine couplings. Comparison of ENDOR spectral features with those of Chla.+ in vitro indicates similar values for Chl1.+ and Chl2.+ radicals but an apparent reduction in unpaired electron spin density for P680.+. It has been proposed from the more detailed studies of purple bacterial reaction centers that such a reduction in spin density can be interpreted as a delocalization over two Chl a molecules. Our calculations therefore suggest that P680.+ is a weakly coupled chlorophyll pair with 82% of the unpaired electron spin located on one chlorophyll of the pair at 15 K. Environmental or geometrical changes to the chlorin ring structure to give a novel monomeric primary donor are also possible.(ABSTRACT TRUNCATED AT 250 WORDS)

ESR and electron nuclear double resonance characterization of the cholesterol oxidase from Brevibacterium sterolicum in its semiquinone state.

Reduction of the flavin of cholesterol oxidase from Brevibacterium sterolicum, at pH values above 7, by sodium dithionite or light irradiation in the presence of EDTA (either in the presence or absence of deazariboflavin) was found to occur through a stable intermediate state. This intermediate had an optical spectrum characteristic of a flavin anionic semiquinone. The rate and extent of reduction were pH-dependent. No semiquinone intermediate was detected during reduction by these agents at pH values of 6.5 or below or at any pH when dehydroisoandrosterone, a protein substrate analogue, was used as reductant. No intermediate radical was detected during the reoxidation process. Treatment of cholesterol oxidase semiquinone with dehydroisoandrosterone did not convert the semiquinone intermediate to the fully reduced state. The absorption coefficient of oxidised cholesterol oxidase at 470 nm is 10.3 M-1 cm-1. The ESR signal of Brevibacterium sterolicum cholesterol oxidase semiquinone is centred at g = 2.004. The linewidth of the signal was 1.48 mT when the protein was studied in H2O or D2O. These data are in agreement with those reported for anionic semiquinones. The linewidths were the same when measured either at X-band or at S-band frequencies, indicating that line broadening is due to hyperfine interactions. The linewidth decreased to 1.43 mT when the substrate, dehydroisoandrosterone, was added. Electron nuclear double resonance (ENDOR) spectroscopy of cholesterol oxidase semiquinone provided further information about the interactions of the flavin radical with protons. A group of signals, with couplings of 9-12 MHz, is attributed to protons on 8-CH3 (Aiso = 10.9 MHz) and on C6 (Aiso = 9 MHz) of the flavin ring. No change in these hyperfine coupling constants was detected when the protein was studied in D2O. However, the hyperfine coupling constant attributed to protons on 8-CH3 decreased by 0.98 MHz when the ENDOR spectrum of the cholesterol oxidase semiquinone was studied in the presence of dehydroisoandrosterone (Aiso = 9.92 MHz). A second group of signals was observed with hyperfine couplings less than 2.5 MHz. Some of these weak couplings disappeared when the protein was transferred to D2O, or when the substrate, dehydroisoandrosterone, was present. These signals are attributed to displaced water protons, or to exchangeable protons from amino-acid residues on the protein near the flavin binding site, involved in substrate stabilisation.

Enhanced diagnostic EPR and ENDOR spectroscopy of radical spin, adducts of deuterated α‐phenyl N‐tert‐butyl nitrone

AbstractDeuterated analogues of C‐phenyl N‐tert‐butyl nitrone (PBN) were synthesized to provide significant gains in spectral sensitivity and resolution in electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) applications. Three deuterated α‐phenyl N‐tert‐butyl nitrones (PBNs) were prepared. EPR spectra of the corresponding radical spin adducts with the phenyl ring (PBN‐d5‐R˙) or tert‐butyl moiety (PBN‐d9‐R˙) deuterated were found to enhance disclosure of the structure of the added radical. The most dramatic increases in EPR resolution, however, were not realized until both the phenyl and tert‐butyl groups were deuterated (PBN‐d14‐R˙). Here, baseline resolution of unique long‐range (e.g. γ‐ and δ‐) hyperfine splittings from the radical addend could be displayed. Representative radical spin adducts of PBN‐d14 (methyl, hydroxyl, aminyl, cyanyl, carbamoyl, and vinyl) were prepared and compared with those of PBN to illustrate this point. It is also shown that when even higher spin adduct resolution is desired the combination of spin trap deuteration and ENDOR may be applied to advantage.

The dark stable tyrosine radical of photosystem 2 studied in three species using ENDOR and EPR spectroscopies.

The dark stable neutral tyrosine radical YD. of photosystem 2 (PS2) has been studied using electron nuclear double-resonance (ENDOR) and electron paramagnetic resonance (EPR) spectroscopies. The proton hyperfine coupling constants of all four ring protons and both beta-methylene protons have been determined for YD. in three species covering the range of oxygenic organisms; a higher plant (spinach), an alga (Chlamydomonas reinhardtii), and a cyanobacterium (Phormidium laminosum). It has generally been assumed that the properties of Yd. are the same in all oxygenic organisms, while in fact there are small but significant differences. The beta-proton coupling constants are shown to be species dependent while the ring proton coupling constants are not. Estimation of the electron spin density distribution of Yd. from all three organisms has been done. This shows that changes in beta-proton coupling constants in each organism arise from the slightly different orientation of the tyrosine ring, relative to the beta-protons. The electron spin density distribution within the tyrosine ring is organism independent. The variations in the beta-proton coupling constants are reflected in the corresponding EPR spectra, where small variations in line width have been detected. These data delineate the range of natural variation in the spectroscopic properties of YD., and by assigning the features of the ENDOR spectrum, provide a basis for both the unification of studies of YD. in different organisms and the study of YZ.. The results are discussed in relation to data in the recent study (Hoganson & Babcock, 1992) using YD. in the cyanobacterium, Synechocystis PCC 6803.

Structure and dynamics of radicals in solids by EPR and ENDOR spectroscopies

EPR and ENDOR (Electron Nuclear DOuble Resonance) spectroscopies give complementary information in the study of paramagnetic species in solids. The ENDOR technique allows the determination of hyperfine tensors an order of magnitude smaller with respect to the EPR one. The molecular motions in solids affect the relaxation of both the transverse and longitudinal magnetization of the paramagnetic probe. The measurement of the transverse relaxation time is traditionally available by the EPR lineshape analysis. Population variations due to the longitudinal relaxation processes can be monitored by the study of the amplitude of the ENDOR spectra.

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

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

Aconitase, a two‐faced protein: enzyme and iron regulatory factor 1 2

In this brief survey, the path of development of our knowledge of the iron-sulfur enzyme aconitase [citrate(isocitrate)hydrolyase EC4.2.1.3.] is traced from its discovery in 1937. Particular emphasis is on developments in the past decade, when EPR, Mössbauer and electron nuclear double resonance spectroscopies, X-ray crystallography, and mutational analysis were applied to the problem. More recently discovered was the significant amino acid sequence identity between mitochondrial aconitase and the iron regulatory factor or iron-responsive element binding protein (IRE-BP). This has led to the realization that IRE-BP is an alternative form of cytosolic (not of mitochondrial) aconitase that is devoid of its cubane Fe-S cluster.

Sum Combination Harmonics in Four-Pulse ESEEM Spectra. Study of the Ligand Geometry in Aqua-Vanadyl Complexes in Polycrystalline and Glass Matrices

Structural differences in the vanadyl pentaaqua complex in two different matrices, a frozen glassy water solution and a polycrystalline Tutton salt, were investigated using the recently designed one dimensional four-pulse electron spin-echo envelope modulation (ESEEM) experiment. The four-pulse ESEEM spectrum consists of the basic nuclear frequencies, να and νβ, of the ligands′ protons along with their sum combination harmonics, (να + νβ). This particular example demonstrates the potential of this new experiment which lies in the generation of highly resolved combination harmonics from which information regarding the hyperfine and quadrupole interactions can be derived. While the combination lines in the two-pulse ESEEM spectra of the polycrystalline sample suffered from low resolution due to a relatively short T2, highly resolved peaks were observed in the four-pulse ESEEM spectra since in this experiment T1 dominates the echo decay rather than T2. Orientation-selective four-pulse ESEEM experiments were performed under conditions that optimize the intensities of the sum combination harmonics. Aqua complexes with H2O and D2O were studied and clear differences in the Fourier transform ESEEM spectra of the two matrices, not detected previously by electron-nuclear double-resonance spectroscopy or two-pulse ESEEM, were evident. The analysis of the spectra was done by simulating the FT-ESEEM spectra of both the H2O and the D2O complexes. For this purpose, an analytical expression describing the four-pulse ESEEM for I = 1 was derived, taking into account the 2H quadrupole interaction as a first-order correction to the nuclear frequencies. The simulations showed that the positions and orientations of the ligands in VO2+(H2O)5 and VO2+(D2O)5 are somewhat different in the frozen glassy solution and in the crystalline matrix of the Tutton salt.

ESR and ENDOR of Cu 2+ in Nafion solutions and in reconstituted membranes

Electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) spectroscopies have been applied to the study of the local environment of paramagnetic counterions in solutions of Nafion perfluorinated membranes in various solvents, and in membranes reconstituted from solutions by evaporation of the solvents at different temperatures. Improved resolution in the ESR and ENDOR spectra was possible by using selectively deuterated solvents and Cu 2+ ions enriched with 63Cu. In contrast to the preferential salvation of the counterions by water in membranes swollen by a water—alcohol mixed solvent, the counterions in Nafion dissolved in the mixed solvent are preferentially solvated by the alcohol. This conclusion is based on the analysis of the solvent-specific ESR parameters and is strengthened by the detection of splittings due to the methanol protons in the ENDOR experiments. The ESR parameters measured in Naflon solutions containing N-methylformamide (NMF) can be explained by assuming 14N ligands around the counterion. These results also suggest that NMF is retained in the reconstituted films obtained by drying ionomer solutions at or below 373 K; drying at 493 K, however, restores the original membrane. The effectiveness of the mixed solvent (water—ethanol) for solubilization of the Naflon membranes appears to be due to preferential salvation of the sulfonic groups by water, and of the perfluorinated chains by methanol or ethanol.

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

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

Synthesis of conjugated polyene carbonyl derivatives of nitroxyl spin-labels and determination of their molecular structure and conformation by electron nuclear double resonance

Formic, propenoic, and pentadienoic acid derivatives of the nitroxyl spin-label 2,2,5,5-tetramethyl-1-oxypyrroline and their corresponding ethyl ester and aldehyde analogues have been synthesized and characterized by chemical methods. Because the π-bonding orbitals of the olefinic side chains are conjugated to the vinyl group of the oxypyrrolinyl ring, these spin-labeled derivatives are excellent spectrophotometic probes, exhibiting high molecular absorption extinction in the near ultraviolet. The molecular structures and conformations of these spin-labeled derivatives have been determined by electron nuclear double resonance (ENDOR) spectroscopy and molecular modeling. Conformational analysis based on torsion angle search calculations, constrained by ENDOR-determined electron-proton separations, showed that the polyene chain has an all planar, trans conformation. In the aldehydes the conformation of the C=O group with respect to the alkene chain is found to be s-trans, while in the acids and esters the -OH and -OC 2 H 5 groups were found to be s-trans, placing the C=O group in an s-cis conformation. The different conformations of the carbonyl group in these compounds are explained on the basis of resonance energy and dipolar interactions of the polyene side chain and the carbonyl or carboxyl group

ChemInform Abstract: An Electron Magnetic Resonance Study on the Photoionization of N‐ Alkylphenothiazines in Dioctadecyldimethylammonium Chloride Frozen Vesicles: The Effect of Urea, 1,3‐Dimethylurea, 1,3‐Diethylurea, and 1, 1′,3,3′‐Tetramethylurea.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ESR and ENDOR relaxation study of molecular motions of a ketone/urea inclusion compound

The radical obtained by γ irradiation of the 10-nonadecanone/urea inclusion compound has been studied by electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy. The EPR line widths have been analyzed, and the characteristics and the rates of the molecular motions of the included molecule have been obtained in the temperature range 160-290 K. Two different types of motion have been detected: the wobbling motion of the methylene groups about the carbon-carbon bond modulates the β proton isotropic hyperfine interactions, while overall uniaxial rotation inside the host channel mainly modulates the anisotropic dipolar interaction of the α proton

7] Continuous wave electron nuclear double resonance spectroscopy

Publisher Summary The rapidly accelerating progress in the biological applications of electron nuclear double resonance (ENDOR) spectroscopy seen since the 1970s reflects a combination of instrumental and conceptual advances. Structural information and its functional implications have been wrested from ENDOR studies of almost all magnetic nuclei coupled to the paramagnetic centers encountered in biological molecules. ENDOR spectroscopy, with the detailed insight it offers into the local structure of a paramagnetic centers in solution and the alterations of such structure during biological function, can often complement the tremendous power of X-ray crystallography in studies of metalloproteins. Interpretation of ENDOR spectra merges intuition with analytic rigor with the limitations of the one, often surmounted by the power of the other. Sensitivity problems persist but are yielding to studies at ever higher frequencies. Fruitful exploitation of Triple resonance in studying metalloproteins has yet to be accomplished. The wide availability of the personal computer, with its dramatically increasing speed and power, has enormously facilitated processing and interpretation of spectroscopic data. A persistent difficulty is to achieve understanding of the relaxation processes and pathways governing ENDOR spectroscopy; such understanding has yet to attain the peaks achieved by other, perhaps simpler, spectoscopies. The ingenuity and resourcefulness of experimenters remain the most powerful tools in the armamentarium of ENDOR spectroscopy.

Influence of cation, solvent and temperature on Z–E isomerisation of radical ion pairs derived from (Z)- and (E)-1,2-bis(2,4,6-trimethylbenzoyl)ethenes by chemical reduction with alkali metals and magnesium. A structural, electron paramagnetic resonance, ENDOR and TRIPLE resonance study

The chemical reduction of (Z)-1,2-bis(2,4,6-trimethylbenzoyl)ethene, 1, and (E)-1,2-bis(2,4,6-trimethylbenzoyl)-ethene, 2, with alkali metals and magnesium amalgam in ethereal solvents has been studied by electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR) and TRIPLE resonance spectroscopies. The structure of the radical ion pairs obtained from 1 exhibits strong dependence upon cation size, solvent polarity and temperature. It was found that (Z)-isomeric ion pairs 1˙– M+ are favoured under conditions involving more ion association, for example, in systems with small counterions, low solvating power of the solvent and at high temperatures. Z–E isomerization occurred at temperatures as low as 193 K even for lithium ion pairs in 1,2-dimethoxyethane. The resulting (E)-isomers decayed significantly with increasing temperature, yielding secondary reduction products. The final paramagnetic species, referred to as D in the text, has strikingly distinct hyperfine data which could be ascribed to a structurally different ion pair resulting from intramolecular cyclization that may occur in the (E)-exo-endo isomeric ion pair. The arguments in favour of such a structure are corroborated by INDO calculations.

ESR and ENDOR of copper(II) complexes with nitrogen donors: probing parameters for prosthetic group modeling of superoxide dismutase

The coordination properties of square-planar complexes of Cu(II) with N 4 or N 2 O 2 ligation using imidazole, pyridine, glycine, ethylenediamine, diethylenetriamineimidazole, and ammonia as ligands were investigated by electron spin resonance (ESR) and electron nuclear double resonance (ENDOR) spectroscopies at 77 K and about 5 K, resistively. Combining isotopic substitution in metal, ligand, and solvent with ESR measurements at X- (9.5 GHz) and Q-band (34 GHz) and second-order spectra simulation, a comprehensive set of ESR parameters was obtained

An electron magnetic resonance study on the photoionization of N-alkylphenothiazines in dioctadecyldimethylammonium chloride frozen vesicles: the effect of urea, 1,3-dimethylurea, 1,3-diethylurea, and 1,1',3,3'-tetramethylurea

Photoionization of N-alkylphenothiazines was carried out in frozen dioctadecyldimethylammonium chloride (DODAC) vesicle suspensions to which various concentrations of urea, 1,3-dimethylurea (DMU), 1,3-diethylurea (DEU), 1,1{prime},3,3{prime}-tetramethylurea (TMU) had been added. The photoproduced cation radical yields were investigated by electron spin resonance (ESR). Electron spin echo modulation (ESEM) and electron nuclear double resonance (ENDOR) spectroscopies were used to probe the microenvironment of the photoproduced cation. The concentration of each of the urea derivatives was systematically varied. Urea, DMU, and DEU showed a similar trend in the photoyield versus concentration, with the highest yield obtained at 1 M additive. For TMU the photoyield of phenothiazine increased as a function of additive concentration. The different photoyield trends are explained in terms of the effect of urea on the DODAC vesicule interface. This interpretation is supported by the ESEM and ENDOR studies. Urea and its derivatives appear to interact with water through the {open_quotes}direct mechanism{close_quotes}. The ESR data for these species suggest that impregnation for the vesicle interface with urea decreases the strength of the photoproduced cation-water interactions which leads to a decrease in the photoyield. The ESE and ENDOR results for TMU suggest that this bulky additive opens up the vesicle interface to water penetration.more » The steady increase in the photoyield with TMU concentration together with the ESEM and ENDOR results support this interpretation of the ESR data. 49 refs., 10 figs.« less