X-ray absorption spectroscopy and EPR studies of oriented spinach thylakoid preparations

Abstract

In this study, oriented Photosystem 11 (PS n) particles from spinach chloroplasts are studied with electron paramagnetic resonance (EPR) and x-ray absorption spectroscopy VAS) to determine more details of the structure of the oxygen evolving complex (OEC).The nature of halide binding to Mn is also studied with C1 K-edge and Mn EXAFS (extended x-ray absorption fine structure) of Mn-C1 model compounds, and with Mn EXAFS of oriented PS 1T in which Br has replaced C1.In chapter 2, oriented PS 11 particles from spinach were studied with EPR spectroscopy. The mosaic spread for these samples was determined by comparison of the oxidized cytochrome b559 (cyt b559+) signal measured at angles of 0" to 360" between the EPR magnetic field and sample membrane normal with simulations of oriented cyt b559+.The tyrosine YD+ signal was measured for these oriented samples at 0" and 90°, and a linear relationship was found between the dichroism found in the tyrosine YD+ signal and the mosaic spread.In chapters 3 and 4, Mn XAS was performed on oriented PS 11 membrane particles isolated from spinach. These studies were on control samples (chapter 3) and on samples treated with ammonia (chapter 4), a water analog which can yield insight into the binding of water by the OEC. Structural features of the tetranuclear Mn cluster and the orientation of the cluster with respect to the lipid bilayer were determined in the S2 state of the Kok cycle 1 for control samples, and on the annealed S2 state for the ammonia-treated samples.Variation of the sample orientation with respect to the x-ray e-vector yields highly dichroic In chapter 5, the binding of halide to Mn was studied using C1 K-edge XAS and Mn EXAFS on Mn-CI model compounds, and Mn EXAFS on oriented Br-treated PS IL From the C1 K-edges of model compounds, direct ligation of C1 to Mn could be confirmed with the presence of a pre-edge feature: 'a forbidden 1s to 3d transition which is seen due to mixing of Mn 3d orbitals with C13p orbitals. Bridging and terminal C1 bonds to Mn could be distinguish& EXAFS of Mn-C1 model compounds revealed that Mn-C1 interactions, especially bridging interactions, make an important contribution to the EXAFS. EXAFS of oriented Br-treated PS 11 showed differences from EXAFS of control CI-treated PS II that were on the same order as the variation from sample to sample. Fits to Br-treated and Cl control samples were also inconclusive. Thus, it is not possible to 9 establish or rule out direct ligation of C1 to Mn in the OEC with these studies.2

Orientation of Calcium in the Mn4Ca Cluster of the Oxygen-Evolving Complex Determined Using Polarized Strontium EXAFS of Photosystem II Membranes

“…For simplicity the dichroic ratio of the ∼3260 G feature of the tyrosine Y D ox (signal II "slow") EPR spectra (24,70) at two extreme angles (0°and 90°) between the sample face and the applied magnetic field (for details see Figure S3) is used to determine the mosaic spread. This method is based on the previous finding that this ratio is inversely related (72) to the mosaic spread as measured from the angle dependence of the cyt b 559 EPR spectra, following the protocol of Blum et al (69). In addition, we confirmed that with the slightly different method of Dau et al (58) the numbers are practically identical (difference ∼5% in the determination of the coordination numbers).…”

“…However, it is cumbersome to collect EPR spectra of cyt b 559 in several orientations from each sample. We devised a simpler method of correlating the dichroism in the EPR signal from the tyrosine Y D ox (24,70,71), which is much easier to collect, to that determined from cyt b 559 (31,58,69,72). X-band (9.2 GHz) EPR spectra of the signal II region (near g = 2.00) were taken for layered samples oriented 0° and 90° in the magnetic field at 20 K and 50 µW power with 2.5 G modulation amplitude.…”

“…The sample face was aligned either parallel (0°) or perpendicular (90°) to the external magnetic field. The dichroic ratio (defined here as the amplitude at 90°d ivided by that at 0°) was taken at ~3260 G. This dichroic ratio ("signal II ratio") was related to the dichroism in the cytochrome b 559 EPR signal (24,31,58,69,70,73) to obtain the mosaic spread (Ω), or half-width of the Gaussian distribution of a particular vector in PS II samples (72). On the basis of previous work from this laboratory (72), a calibration line was constructed, equating mosaic spread to a measured signal II ratio.…”

Removal of Ca2+ from the Oxygen-Evolving Complex in Photosystem II Has Minimal Effect on the Mn4O5 Core Structure: A Polarized Mn X-ray Absorption Spectroscopy Study

“…The dichroic ratio of the oriented samples used for XAS experiments ranged from 3.3 to 5.3 with an average of 3.9 ± 0.6. According to the calibration curve of Andrews, 56 which relates the dichroic ratio to Ω via the angular dependence of the cytochrome b 559 EPR signal, the mosaic spread was found to be 15–20°. For evaluation of the angle dependence of the apparent coordination numbers N app, j , derived from polarized EXAFS data (sections 2.5 and 3.5), 30 the lower limit of 20° was chosen.…”

“…The temperature was 8 K, the microwave power 0.5 mW and the modulation amplitude 32 G. None of the batches of Ca 2+ -depleted and Ca 2+ -reconstituted samples contained detectable amounts of Mn II . In order to determine the mosaic spread Ω as a measure for the degree of disorder (Section S2 of the Supporting Information), EPR spectra of Y D • (D2-Tyr160) at g = 2 were collected from one-dimensionally ordered samples at orientations of 0° and 90° relative to the magnetic field at 20 K and 25 μW microwave power with 2.5 G modulation amplitude. , Spectra of the respective Mn 4 O 5 Ca cluster signals were recorded for each sample at 8 K with 30 mW microwave power and 32 G modulation amplitude. The various MLS spectra were background-corrected by light-minus-dark subtraction (i.e., the difference between the spectra after and before sample illumination).…”