Proton Matrix ENDOR Research Articles

Proton Matrix ENDOR Studies on Ca2+-depleted and Sr2+-substituted Manganese Cluster in Photosystem II

Proton matrix ENDOR spectra were measured for Ca(2+)-depleted and Sr(2+)-substituted photosystem II (PSII) membrane samples from spinach and core complexes from Thermosynechococcus vulcanus in the S2 state. The ENDOR spectra obtained were similar for untreated PSII from T. vulcanus and spinach, as well as for Ca(2+)-containing and Sr(2+)-substituted PSII, indicating that the proton arrangements around the manganese cluster in cyanobacterial and higher plant PSII and Ca(2+)-containing and Sr(2+)-substituted PSII are similar in the S2 state, in agreement with the similarity of the crystal structure of both Ca(2+)-containing and Sr(2+)-substituted PSII in the S1 state. Nevertheless, slightly different hyperfine separations were found between Ca(2+)-containing and Sr(2+)-substituted PSII because of modifications of the water protons ligating to the Sr(2+) ion. Importantly, Ca(2+) depletion caused the loss of ENDOR signals with a 1.36-MHz separation because of the loss of the water proton W4 connecting Ca(2+) and YZ directly. With respect to the crystal structure and the functions of Ca(2+) in oxygen evolution, it was concluded that the roles of Ca(2+) and Sr(2+) involve the maintenance of the hydrogen bond network near the Ca(2+) site and electron transfer pathway to the manganese cluster.

Highly resolved proton matrix ENDOR of oriented photosystem II membranes in the S2 state

Proton matrix ENDOR was performed to investigate the protons close to the manganese cluster in oriented samples of photosystem II (PS II). Eight pairs of ENDOR signals were detected in oriented PS II membranes. At an angle of θ=0° between the membrane normal vector n and the external field H0, five pairs of ENDOR signals were exchangeable in D2O medium and three pairs were not exchangeable in D2O medium. The hyperfine splitting of 3.60MHz at θ=0° increased to 3.80MHz at θ=90°. The non-exchangeable signals with 1.73MHz hyperfine splitting at θ=0°, which were assigned to a proton in an amino acid residue, were not detected at θ=90° in oriented PS II or in non-oriented PS II. Highly resolved spectra show that only limited numbers of protons were detected by CW-ENDOR spectra, although many protons were located near the CaMn4O5 cluster. The detected exchangeable protons were proposed to arise from the protons belonging to the water molecules, labeled W1-W4 in the 1.9Å crystal structure, directly ligated to the CaMn4O5 cluster, and nearby amino-acid residue.

3D0900 膜配向したPS IIのENDOR法によるMnクラスター周辺のプロトンの位置の決定(光生物-光合成,口頭発表,日本生物物理学会第50回年会(2012年度))

3D0900 膜配向したPS IIのENDOR法によるMnクラスター周辺のプロトンの位置の決定(光生物-光合成,口頭発表,日本生物物理学会第50回年会(2012年度))

Protons bound to the Mn cluster in photosystem II oxygen evolving complex detected by proton matrix ENDOR

Protons in the vicinity of the oxygen-evolving manganese cluster in photosystem II were studied by proton matrix ENDOR. Six pairs of proton ENDOR signals were detected in both the S 0 and S 2 states of the Mn-cluster. Two pairs of signals that show hyperfine constants of 2.3/2.2 and 4.0 MHz, respectively, disappeared after D 2O incubation in both states. The signals with 2.3/2.2 MHz hyperfine constants in S 0 and S 2 state multiline disappeared after 3 h of D 2O incubation in the S 0 and S 1 states, respectively. The signal with 4.0 MHz hyperfine constants in S 0 state multiline disappeared after 3 h of D 2O incubation in the S 0 state, while the similar signal in S 2 state multiline disappeared only after 24 h of D 2O incubation in the S 1 state. The different proton exchange rates seem to be ascribable to the change in affinities of water molecules to the variation in oxidation state of the Mn cluster during the water oxidation cycle. Based on the point dipole approximation, the distances between the center of electronic spin of the Mn cluster and the exchangeable protons were estimated to be 3.3/3.2 and 2.7 Å, respectively. These short distances suggest the protons belong to the water molecules ligated to the manganese cluster. We propose a model for the binding of water to the manganese cluster based on these results.

Determination of distances from tyrosine D to Q A and chlorophyll Z in photosystem II studied by `2+1' pulsed EPR

A `2+1' pulse sequence electron spin echo (ESE) method was applied to measure the dipole interactions between the tyrosine Y D + and Q A − in Photosystem II (PS II). In a CN −-treated PS II, Q A − EPR signal was observed at g=2.0045 position, because the non-heme Fe(II) was converted into a low-spin ( S=0) state. The radical pair of Y D +Q A − was trapped by illumination for 8 min at 273 K, followed by dark adaptation for 3 min and freezing into 77 K. By using a proton matrix ENDOR, these trapped radicals were confirmed to be Y D + and Q A −, respectively. The distance between the radical pair was estimated from the dipole interaction constant fitted to the observed `2+1' ESE time profile. The distance of Y D +–Q A − is determined to be 38.8±1.1 Å. The magnetic dipole interaction between Y D + and Chl Z + was determined in a Tris-treated PS II in which Chl Z + was generated by illumination at 200 K for 10 min. The Y D +–Chl Z + distance was estimated to be 29.4±0.5 Å.

Microenvironments of tyrosine D + and tyrosine Z + in Photosystem II studied by proton matrix ENDOR

Proton matrix ENDOR spectra of tyrosines Z +(Y + Z) and D +(Y + D) were studied in the frozen Tris-treated PS II. ENDOR spectra of the tyrosines in oriented membranes observed at 10 K have shown similar angular dependence with similar line pair separations, suggesting that the local arrangement of protons surrounding both radical species is similar. The angular dependence of EPR spectra showed the C-O bonds in Y + Z and Y + D lie parallel to the membrane normal. With increasing temperature, the intensity of the line pair with 1.2 MHz separation decreased above 25 K for Y + Z, and above 110 K for Y + D, respectively. Assuming a motion of the proton responsible to the line pair, the activation energies for the motional broadening were estimated to be 86 ± 20 J/mol for Y + Z, and 810 ± 200 J/mol for Y + D, respectively. The broadening may be ascribed to small amplitude vibrations of protons surrounding tyrosine radicals. The intensities for the peaks with about 7 MHz separation assigned to the OH proton of the tyrosine decreased, which was ascribed to a similar motional broadening. The difference in temperature dependence of line broadening of the two radicals suggests that the functional difference between them may be ascribable to differences in flexibility in their microenvironment.

Matrix ENDOR of tyrosine D + in oriented Photosystem II membranes

Proton matrix ENDOR spectroscopy has been used to probe the proton environment of the stable tyrosine D + radical present in Photosystem II membranes. ENDOR spectra in randomly oriented and oriented membranes are reported. Simulations of the spectra are given using both the point dipole approximation and the spin distribution over the tyrosine radical, which were found to provide reasonably good fits. The following results are reported: (1) Five groups of protons can be detected in the matrix ENDOR spectra of D +, that have ENDOR peak separations less than 2 MHz, and are located at distances between 3.5 and 6.7 Å from the center of the tyrosine ring. These protons are directed approximately parallel to the PS II membrane plane. (2) Three pairs of ENDOR lines with separations of 2.9, 4.1 and 4.7 MHz, are also reported, together with a signal from the hydrogen bonded to the terminal oxygen in the tyrosine molecule. These protons can be exchanged by treatment in a deuterated buffer. From the orientation dependence of these ENDOR lines, it is concluded that they correspond to protons with distance vectors from the ring center that are perpendicular to the PS II membrane plane. We suggest that this specific organization of protons around the tyrosyl D + radical contributes to the particular orientation of the β-CH 2 group in the radical, and may also play a role in the unusual stability of the D + radical form in the PS II membrane.

Electron spin resonance and proton matrix electron nuclear double resonance studies of N,N,N',N'-tetramethylbenzidine photoionization in sodium and lithium dodecyl sulfate micelles: structural effects of crown ethers

The study of model membrane systems enjoys increasing attention within the area of solar energy research. An electron nuclear double resonance and electron spin resonance study of photogenerated N,N,N[prime],N[prime]-tetramethylbenzidine (TMB) cation in frozen suspensions of lithium (LDS) and sodium (SDS) dodecyl sulfate micelles containing various concentrations of cyclic polyethers was undertaken. The relative location of the TMB cation within the organic aggregate was determined from the proton matrix ENDOR line width at 142 K. A broader line width was observed in LDS compared to SDS micelles, which is due to the fact that the larger lithium cation opens the micellar interface resulting in increased hydration and deeper solubilization of TMB. The proton matrix ENDOR line width decreased upon addition of crown ethers. This decrease may be explained by displacement of the TMB toward the interface as a result of the decrease in ionic strength caused by the complexation of the countercations. The photoyield shows a slight increase with addition of crown ethers. This increase is most likely caused by the increase in the effective anionic charge of the micelle effected by the complexation of the sodium or lithium ions by the crown ethers. This increase in the anionic charge mitigatesmore » the rate of thermal back electron transfer resulting in an increased photoyield. 54 refs., 6 figs., 2 tabs.« less

E.p.r. and ENDOR studies of Argonne Premium coals doped with nitroxide spin probes

E.p.r. and ENDOR measurements of five Argonne Premium Coal Samples (APCS) varying in rank from low-volatile (Pocahontas, No. 5) to lignite (Beulah-Zap, No. 8) either undoped or doped with nitroxide spin probes and a series of nitroxide radicals are reported. For the first time proton hyperfine coupling constants have been deduced from ENDOR studies of tempamine, DCT-tempamine and a carboxaldimine nitroxide radical in toluene solution at 185 K. Proton matrix ENDOR signals at the free proton frequency and a broad ENDOR signal were observed for all undoped APCS, except for a sample of Beulah-Zap in which no proton ENDOR lines were observed. For the spin probe doped Beulah-Zap, Blind Canyon and Illinois No. 6, the ENDOR spectrum consisted of the lines observed for the undoped coal, and two pairs of proton ENDOR lines with hyperfine couplings of 3.6 MHz and 0.18 MHz. The lines with the coupling of 3.6 MHz are due to nitroxide radicals, assigned by similarity to the ENDOR results of the nitroxide spin probes in toluene solution at 120 K. The existence of the second pair of lines with hyperfine coupling constant 0.18 MHz was assumed to be due to the interaction of surrounding matrix coal protons with the nitroxide species, substantiating an earlier report of hydrogen bonding to the nitroxide spin probe.

ENDOR study on the position of hydrogens close to the manganese cluster in S 2 state of photosystem II

Proton matrix ENDOR of the manganese multiline in the S 2, state of photosystem II membranes from spinach has been investigated. The spectral structure over a range of frequencies of ± 2 MHz centered on the position for a free proton was analyzed through employing a simulation method based on the dipolar interaction between electron and proton magnetic moments. The 6 pairs of lines resolved were attributed to the surrounding proton populations at distances varying within the range 2.7–6.0 Å from the putative manganese center. Two of the 6 pairs, namely those corresponding to protons at distances of 2.7 and 3.2 Å from the manganese center were eliminated on washing of the membranes with deuterated buffer. This suggests that these protons belong to the water molecules coordinating to the manganese cluster. The presence of ethylene glycol instead of glycerol and sucrose in the buffer broadened both EPR and ENDOR spectra. This suggests that ethylene glycol molecules are also accessible to the two-manganese cluster. A possible model of water association to the manganese ions in the photosynthetic water-oxidation system is proposed.

Matrix ENDOR of polyenyl radicals in polymers

AbstractEPR and matrix ENDOR spectra have been examined for polyenyl radicals in γ‐irradiated PVF, PVF2, PVC, and PMMA polymers. Proton matrix ENDOR is observed for all four polymers, and fluorine matrix ENDOR for PVF and PVF2. By line shape analysis of the ENDOR spectra obtained under comparable conditions, delocalization diameters for the unpaired electron of the polyenyl radical in each polymer are obtained. These diameters indicate extensive delocalization over 5–7 carbon double bonds for the polyenyl radicals investigated. It is suggested that these conjugated and crosslinked radiation products account for the observed nondevelopment of electron beam resist PMMA material at high radiation charge densities.