Structure and Orientation of the Mn4Ca Cluster in Plant Photosystem II Membranes Studied by Polarized Range-extended X-ray Absorption Spectroscopy
2006; Elsevier BV; Volume: 282; Issue: 10 Linguagem: Inglês
10.1074/jbc.m610505200
ISSN1083-351X
AutoresYulia Pushkar, Junko Yano, Pieter Glatzel, Johannes Messinger, A.K.De K. Lewis, Kenneth Sauer, Uwe Bergmann, Vittal K. Yachandra,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoX-ray absorption spectroscopy has provided important insights into the structure and function of the Mn4Ca cluster in the oxygen-evolving complex of Photosystem II (PS II). The range of manganese extended x-ray absorption fine structure data collected from PS II until now has been, however, limited by the presence of iron in PS II. Using a crystal spectrometer with high energy resolution to detect solely the manganese Kα fluorescence, we are able to extend the extended x-ray absorption fine structure range beyond the onset of the iron absorption edge. This results in improvement in resolution of the manganese-backscatterer distances in PS II from 0.14 to 0.09Ä. The high resolution data obtained from oriented spinach PS II membranes in the S1 state show that there are three di-μ-oxo-bridged manganese-manganese distances of ∼2.7 and ∼2.8Ä in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of ∼60° relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the ∼3.2Ä manganese-manganese and the ∼3.4Ä manganese-calcium interactions in oriented PS II samples and determine their orientation relative to the membrane normal. The average of the manganese-calcium vectors at ∼3.4Ä is aligned along the membrane normal, while the ∼3.2Ä manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn4Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models. X-ray absorption spectroscopy has provided important insights into the structure and function of the Mn4Ca cluster in the oxygen-evolving complex of Photosystem II (PS II). The range of manganese extended x-ray absorption fine structure data collected from PS II until now has been, however, limited by the presence of iron in PS II. Using a crystal spectrometer with high energy resolution to detect solely the manganese Kα fluorescence, we are able to extend the extended x-ray absorption fine structure range beyond the onset of the iron absorption edge. This results in improvement in resolution of the manganese-backscatterer distances in PS II from 0.14 to 0.09Ä. The high resolution data obtained from oriented spinach PS II membranes in the S1 state show that there are three di-μ-oxo-bridged manganese-manganese distances of ∼2.7 and ∼2.8Ä in a 2:1 ratio and that these three manganese-manganese vectors are aligned at an average orientation of ∼60° relative to the membrane normal. Furthermore, we are able to observe the separation of the Fourier peaks corresponding to the ∼3.2Ä manganese-manganese and the ∼3.4Ä manganese-calcium interactions in oriented PS II samples and determine their orientation relative to the membrane normal. The average of the manganese-calcium vectors at ∼3.4Ä is aligned along the membrane normal, while the ∼3.2Ä manganese-manganese vector is oriented near the membrane plane. A comparison of this structural information with the proposed Mn4Ca cluster models based on spectroscopic and diffraction data provides input for refining and selecting among these models. Photosynthesis by green plants, algae, and cyanobacteria provides essentially all of the dioxygen in the biosphere as a byproduct of the electron transfer processes utilizing water as the ultimate electron source: 2H2O → O2 + 4H+ + 4e–. Water oxidation is a light-driven reaction that is catalyzed by an oxygen-evolving complex (OEC) 4The abbreviations used are: OEC, oxygen-evolving complex; EXAFS, extended x-ray absorption fine structure; FT, Fourier transform; PS II, photosystem II; XANES, x-ray absorption near edge spectroscopy; XAS, x-ray absorption spectroscopy; XRD, x-ray diffraction; MES, 4-morpholineethanesulfonic acid. of Photosystem II (PS II) (1Debus R.J. Biochim. Biophys. Acta. 1992; 1102: 269-352Crossref PubMed Scopus (1093) Google Scholar, 2Rutherford A.W. Zimmermann J.-L. Boussac A. Barber J. The Photosystems: Structure, Function, and Molecular Biology. Elsevier Science Publishers B.V., Amsterdam1992: 179-229Crossref Google Scholar, 3Ort D.R. Yocum C.F. Oxygenic Photosynthesis: The Light Reactions. Kluwer Academic Publishers, Dordrecht, The Netherlands1996Crossref Google Scholar, 4Wydrzynski T. Satoh S. Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase. Springer, Dordrecht, The Netherlands2005Crossref Google Scholar). The active site of the OEC is known to be a protein-bound complex containing four manganese and one calcium atom. This complex cycles through a series of five intermediate redox states that are referred to as S states (S0 to S4) (5Kok B. Forbush B. McGloin M. Photochem. Photobiol. 1970; 11: 457-475Crossref PubMed Scopus (1853) Google Scholar). The S state transitions are driven by successive light-induced one-electron oxidations of the PS II reaction center. In each step the complex accumulates oxidizing equivalents until dioxygen is released during the spontaneous return from S4 to S0. Many of the proposed mechanisms of water oxidation depend critically on knowledge of the Mn4Ca cluster structure. To date, structural models of the OEC complex have been suggested based on EPR techniques (6Miller A.F. Brudvig G.W. Biochim. Biophys. Acta. 1991; 1056: 1-18Crossref PubMed Scopus (227) Google Scholar, 7Hasegawa K. Ono T.-a. Inoue Y. Kusunoki M. Chem. Phys. Lett. 1999; 300: 9-19Crossref Scopus (62) Google Scholar, 8Carrell T.G. Tyryshkin A.M. Dismukes G.C. J. Biol. Inorg. Chem. 2002; 7: 2-22Crossref PubMed Scopus (197) Google Scholar, 9Britt R.D. Campbell K.A. Peloquin J.M. Gilchrist M.L. Aznar C.P. Dicus M.M. Robblee J. Messinger J. Biochim. Biophys. Acta. 2004; 1655: 158-171Crossref PubMed Scopus (207) Google Scholar), x-ray absorption spectroscopy (XAS) (10DeRose V.J. Mukerji I. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. J. Am. Chem. Soc. 1994; 116: 5239-5249Crossref Scopus (120) Google Scholar, 11Yachandra V.K. Sauer K. Klein M.P. Chem. Rev. 1996; 96: 2927-2950Crossref PubMed Scopus (960) Google Scholar, 12Penner-Hahn J.E. Struct. Bond. 1998; 90: 1-36Crossref Google Scholar, 13Sauer K. Yano J. Yachandra V.K. Photosynth. Res. 2005; 85: 73-86Crossref PubMed Scopus (46) Google Scholar, 14Yano J. Kern J. Sauer K. Latimer M. Pushkar Y. Biesiadka J. Loll B. Saenger W. Messinger J. Zouni A. Yachandra V.K. Science. 2006; 314: 821-825Crossref PubMed Scopus (705) Google Scholar), x-ray diffraction (XRD) (15Kamiya N. Shen J.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 98-103Crossref PubMed Scopus (1013) Google Scholar, 16Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2904) Google Scholar, 17Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1630) Google Scholar), and infrared spectroscopy (Fourier transform infrared) (18Chu H.-A. Hillier W. Law N.A. Babcock G.T. Biochim. Biophys. Acta. 2001; 1503: 69-82Crossref PubMed Scopus (100) Google Scholar). The XRD studies (3.0–3.8 Ä resolution) have located the Mn4Ca cluster in the density map (16Ferreira K.N. Iverson T.M. Maghlaoui K. Barber J. Iwata S. Science. 2004; 303: 1831-1838Crossref PubMed Scopus (2904) Google Scholar, 17Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1630) Google Scholar) and confirmed the presence of calcium in the OEC cluster, as had been shown previously by EPR (19Boussac A. Rutherford A.W. Chem. Scr. 1988; 28A: 123-126Google Scholar, 20Boussac A. Rutherford A.W. Biochemistry. 1988; 27: 3476-3483Crossref Scopus (229) Google Scholar, 21Boussac A. Zimmermann J.-L. Rutherford A.W. Biochemistry. 1989; 28: 8984-8989Crossref PubMed Scopus (250) Google Scholar) and by extended x-ray absorption fine structure (EXAFS) spectroscopy (22Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E. Sauer K. Yachandra V.K. Biochemistry. 2002; 41: 12928-12933Crossref PubMed Scopus (120) Google Scholar, 23Cinco R.M. Robblee J.H. Messinger J. Fernandez C. Holman K.L.M. Sauer K. Yachandra V.K. Biochemistry. 2004; 43: 13271-13282Crossref PubMed Scopus (53) Google Scholar). A recent XAS study showed that the OEC complex is very susceptible to reduction and disruption during x-ray exposure, under the conditions used in collecting the published XRD data (24Yano J. Kern J. Irrgang K.-D. Latimer M.J. Bergmann U. Glatzel P. Pushkar Y. Biesiadka J. Loll B. Sauer K. Messinger J. Zouni A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12047-12052Crossref PubMed Scopus (552) Google Scholar). Consequently, the precise location of the manganese and calcium atoms has not been reliably established within active OEC centers by XRD, as acknowledged in the most recent study (17Loll B. Kern J. Saenger W. Zouni A. Biesiadka J. Nature. 2005; 438: 1040-1044Crossref PubMed Scopus (1630) Google Scholar). Manganese XAS enables a detailed analysis of the Mn4Ca cluster in the OEC. X-ray absorption near-edge structure (XANES) contains information on the electronic structure and changes in oxidation states of the manganese that accompany S state transitions (25Messinger J. Robblee J.H. Bergmann U. Fernandez C. Glatzel P. Visser H. Cinco R.M. McFarlane K.L. Bellacchio E. Pizarro S.A. Cramer S.P. Sauer K. Klein M.P. Yachandra V.K. J. Am. Chem. Soc. 2001; 123: 7804-7820Crossref PubMed Scopus (259) Google Scholar). EXAFS allows for a precise determination of manganese-backscatterer distances (26Yachandra V.K. Wydrzynski T. Satoh S. Photosystem II: The Light-Driven Water:Plastoquinone Oxidoreductase. Springer, Dordrecht, The Netherlands2005: 235-260Google Scholar) and is, furthermore, very sensitive for establishing the permissible x-ray dose (24Yano J. Kern J. Irrgang K.-D. Latimer M.J. Bergmann U. Glatzel P. Pushkar Y. Biesiadka J. Loll B. Sauer K. Messinger J. Zouni A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12047-12052Crossref PubMed Scopus (552) Google Scholar). Recent EXAFS studies of the Mn4Ca cluster of the OEC have led to the conclusion that there are three manganese-manganese vectors in the range 2.7–2.9 Ä reflecting di-μ-oxo-bridged manganese atom pairs (27Yano J. Pushkar Y. Glatzel P. Lewis A. Sauer K. Messinger J. Bergmann U. Yachandra V.K. J. Am. Chem. Soc. 2005; 127: 14974-14975Crossref PubMed Scopus (171) Google Scholar), one manganese-manganese vector at 3.3 Ä, and one or two manganese-calcium vectors at 3.4 Ä (22Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E. Sauer K. Yachandra V.K. Biochemistry. 2002; 41: 12928-12933Crossref PubMed Scopus (120) Google Scholar, 23Cinco R.M. Robblee J.H. Messinger J. Fernandez C. Holman K.L.M. Sauer K. Yachandra V.K. Biochemistry. 2004; 43: 13271-13282Crossref PubMed Scopus (53) Google Scholar). The ability of the EXAFS technique to determine the presence of similar backscatterers at closely separated distances, ΔR, is dependent on Δk, the width of the k-space EXAFS data set (Ä–1) (for details see supplemental data). The presence of iron, partly an integral component of the OEC and partly from adventitious sources, restricts the useful range in conventional manganese EXAFS to ∼550 eV (Δk = 12.5 Ä–1) above the manganese edge (iron K-edge at 7120 eV). Consequently, in PS II the manganese-manganese distance resolution is limited to Δr = 0.14 Ä, meaning that two manganese-manganese vectors should differ greater than 0.14 Ä to be resolved. Improvement in manganese-backscatterer distance resolution is critical for precise structural and mechanistic studies of the OEC. Conventional EXAFS spectra of PS II samples are based on the detection of the manganese Kα1,2 fluorescence (∼5.9 keV) using a solid state detector of at best 150–200 eV from full width at half-maximum resolution (28Scott R.A. Rousseau D.L. Structural and Resonance Techniques in Biological Research. Academic Press, Orlando, FL1984: 295-362Crossref Google Scholar, 29Cramer S.P. Koningsberger D.C. Prins R. X-ray Absorption: Principles, Applications, Techniques of EXAFS, SEXAFS and XANES. Wiley, New York1988: 257-326Google Scholar, 30Yachandra V.K. Methods Enzymol. 1995; 246: 638-675Crossref PubMed Scopus (53) Google Scholar), making it impossible to discriminate completely against the Fe Kα1,2 fluorescence at 6.4 keV (see Fig. 1). This limitation can be overcome by utilizing a crystal monochromator with high resolution (∼1 eV) for the fluorescence detection (31Bergmann U. Cramer S.P. SPIE Conference on Crystal and Multilayer Optics. SPIE, San Diego, CA1998: 198-209Google Scholar, 32Glatzel P. de Groot F.M.F. Manoilova O. Grandjean D. Weckhuysen B.M. Bergmann U. Barrea R. Phys. Rev. B. 2005; 72: 014-117Crossref Scopus (42) Google Scholar). Recently we showed that manganese EXAFS of the OEC can be collected up to ∼1000 eV (k = 16.1 Ä–1) above the manganese K-edge (27Yano J. Pushkar Y. Glatzel P. Lewis A. Sauer K. Messinger J. Bergmann U. Yachandra V.K. J. Am. Chem. Soc. 2005; 127: 14974-14975Crossref PubMed Scopus (171) Google Scholar), improving the manganese-backscatterer distance resolution to 0.09 Ä. This enabled us to study the heterogeneity in the manganese-manganese distances of solution samples in the S1 and S2 states, providing evidence for three manganese-manganese distances of ∼2.7 and ∼2.8 Ä present in a 2:1 ratio (27Yano J. Pushkar Y. Glatzel P. Lewis A. Sauer K. Messinger J. Bergmann U. Yachandra V.K. J. Am. Chem. Soc. 2005; 127: 14974-14975Crossref PubMed Scopus (171) Google Scholar). These improvements in determining the structural parameters are important for choosing among different proposed structural models, and they provide an opportunity for investigating the changes that occur as the Mn4Ca catalyst cycles through the S states. Additional geometric information about the spatial arrangement of manganese-backscatter vectors can be obtained if oriented PS II samples, such as oriented membranes or single crystals, are used for the measurement of EXAFS dichroism with linearly polarized synchrotron x-rays. Collection of the polarized EXAFS spectra on oriented PS II membranes at different angles between the membrane normal and the x-ray electric field vector results in dichroism that depends on how the particular absorber manganese-backscatter vector is aligned with respect to the electric field of the x-ray beam. Thus, the average orientation of a particular manganese-backscatter vector relative to the membrane normal and the average number of scatterers per absorbing atom can be determined (33George G.N. Prince R.C. Cramer S.P. Science. 1989; 243: 789-791Crossref PubMed Scopus (203) Google Scholar, 34Mukerji I. Andrews J.C. DeRose V.J. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1994; 33: 9712-9721Crossref PubMed Scopus (58) Google Scholar, 35Dau H. Andrews J.C. Roelofs T.A. Latimer M.J. Liang W. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1995; 34: 5274-5287Crossref PubMed Scopus (106) Google Scholar). Previous studies on oriented native and NH3-treated PS II membranes were based on conventional EXAFS. Average angles relative to the membrane normal of ∼60° for the ∼2.7 Ä vectors (di-μ-oxo-bridged Mn2 units) and ∼43° for the ∼3.3 Ä vectors (superposition of mono-μ-oxo-bridged manganese-manganese and manganese-calcium vectors) have been reported in two studies (34Mukerji I. Andrews J.C. DeRose V.J. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1994; 33: 9712-9721Crossref PubMed Scopus (58) Google Scholar, 35Dau H. Andrews J.C. Roelofs T.A. Latimer M.J. Liang W. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1995; 34: 5274-5287Crossref PubMed Scopus (106) Google Scholar), whereas another study reported an average angle of 80 ± 10° for the ∼2.7 Ä vectors without providing results for the ∼3.3 Ä vector (36Schiller H. Dittmer J. Iuzzolino L. Dörner W. Meyer-Klaucke W. Solé V.A. Nolting H.-F. Dau H. Biochemistry. 1998; 37: 7340-7350Crossref PubMed Scopus (62) Google Scholar). Because of the limited resolution, conventional EXAFS is not able to determine the orientations of the individual manganese-manganese and manganese-calcium vectors in the 3.2–3.4 Ä region. In a complementary study, strontium K-edge polarized EXAFS of strontium-reactivated PS II membranes was used to predict the manganese-calcium orientation. It showed a lower and upper limit of 0 and 23°, respectively, for the average angle between the manganese-strontium vector(s) and the membrane normal and yielded an isotropic coordination number of manganese neighbors to strontium of either one or two (23Cinco R.M. Robblee J.H. Messinger J. Fernandez C. Holman K.L.M. Sauer K. Yachandra V.K. Biochemistry. 2004; 43: 13271-13282Crossref PubMed Scopus (53) Google Scholar). A recent polarized x-ray absorption spectroscopy study of PS II single crystals from cyanobacteria, using an x-ray dose below the threshold of damage, has derived feasible structures for the Mn4Ca cluster and the orientation of the cluster in the PS II crystal (14Yano J. Kern J. Sauer K. Latimer M. Pushkar Y. Biesiadka J. Loll B. Saenger W. Messinger J. Zouni A. Yachandra V.K. Science. 2006; 314: 821-825Crossref PubMed Scopus (705) Google Scholar). In this work, we applied range-extended EXAFS to study the dichroism of the Mn4Ca cluster in oriented PS II membranes from spinach chloroplasts. The study shows: (i) the separation of the manganese-manganese (∼3.2 Ä) and manganese-calcium (∼3.4 Ä) vectors, which allows independent analysis of their orientation relative to the membrane normal; (ii) the determination of the dichroism characteristics of the three short manganese-manganese vectors (two at 2.7 Ä and one at 2.8 Ä) and their orientation in the PS II membrane. These results are used to discuss the structure and orientation of the Mn4Ca cluster in the PS II membrane. Sample Preparation and Characterization—PS II samples were prepared from spinach as previously described (37Berthold D.A. Babcock G.T. Yocum C.F. FEBS Lett. 1981; 134: 231-234Crossref Scopus (1662) Google Scholar). They typically contain 4 manganese per 200–250 chlorophylls. The oxygen evolution rates for the PS II samples used in this study are between 400 and 500 μmol O2/(mg chlorophylls · h). The membranes were resuspended in 50 mm MES buffer, pH = 6.0, containing 0.4 m sucrose and 5 mm CaCl2 and pelleted by centrifugation at 4 °C (39,000 × g, 1 h). One or two drops of 50 mm MES buffer were added to the pellet, and the resulting paste was painted onto Mylar tape. The PS II membranes were dried under a stream of cold nitrogen gas at 4 °C in the dark for ∼1 h, as described previously (38Rutherford A.W. Biochim. Biophys. Acta. 1985; 807: 189-201Crossref Scopus (126) Google Scholar). This process was repeated five to seven times to generate samples with a sufficiently thick sample layer for the x-ray absorption experiment. The paint-and-dry cycles produce one-dimensionally ordered samples with a preferred orientation of the PS II membrane normal perpendicular to the substrate surface. The extent of orientation (mosaic spread, which is the half-width of the Gaussian distribution of the angle of the membrane normal to the substrate normal in the PS II samples) was assessed from the angle dependence of the Tyr Dox and cytochrome b559 EPR signals (see supplemental data). X-band EPR spectroscopy was performed with a Varian E-109 spectrometer, a standard TE102 cavity, and an Air Products liquid helium cryostat. The samples used in this study displayed a mosaic spread of 15–20°. After drying the samples, their integrity was assayed by monitoring the amount of S2 multiline signal formed upon sample illumination at 195 K. The amplitude of the manganese signal was the same as that obtained from randomly oriented membranes at a similar concentration. Manganese K-edge XANES spectra of oriented samples can be used to reconstruct the solution XANES spectrum, which is very sensitive to the manganese oxidation state and damaged PS II centers containing Mn2+. The two spectra are indistinguishable, indicating the intact state of the oriented samples. Data Collection—The x-ray spectra were recorded on the BioCAT undulator beamline 18-ID at the Advanced Photon Source (Argonne, IL). The energy of the incident x-rays was selected by means of a nitrogen-cooled silicon double-crystal monochromator at (111) orientation, yielding ∼1-eV resolution. The monochromator energy was calibrated using the preedge peak energy of KMnO4 at 6543.3 eV. Higher harmonics from the monochromator were rejected by the focusing mirror. The incident beam intensity was set to ∼4 × 1012 photons/s (∼60% of the flux available at 18-ID) at a beam size of 1 × 2 mm2. This allowed us to perform fast EXAFS scans in continuous mode before the onset of radiation damage; 15 s per sweep, energy range 6500 to 7500 eV in 1 eV increments, one sweep per spot on sample, 15–20 different spots per sample depending on orientation, ∼100 samples per orientation. The EXAFS scan parameters were chosen subsequent to and on the basis of a radiation damage study of the samples. XANES spectra were collected under identical conditions (number of photons, time and temperature that were used for subsequent EXAFS measurements), and the inflection point energy of the XANES spectra was monitored for any shifts to establish the safe x-ray dose (24Yano J. Kern J. Irrgang K.-D. Latimer M.J. Bergmann U. Glatzel P. Pushkar Y. Biesiadka J. Loll B. Sauer K. Messinger J. Zouni A. Yachandra V.K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 12047-12052Crossref PubMed Scopus (552) Google Scholar). Radiation damage measurements were determined for both 15 and 75° orientations of the samples used in the study and were repeated each time we had x-ray beamtime at the synchrotron sources to account for any changes in the beam characteristics. A second 15-s sweep of the EXAFS for some samples was collected and the spectra were unchanged, providing additional confirmation of the absence of radiation damage. To avoid unnecessary sample exposure, a beam shutter was automatically inserted when data were not being collected. The manganese Kα fluorescence was detected by four spherically bent germanium analyzers (8.9 cm diameter, 85 cm radius of curvature) using the (333) Bragg reflection in a Rowland geometry. The analyzer energy was tuned to the manganese Kα1 peak at 5899 eV at a Bragg angle of 74.84°. A nitrogen-cooled solid state (germanium) detector was placed at the common focus of the four crystals on the intersecting Rowland circles. The analyzer bandwidth of 0.8 eV was determined by measuring the elastically scattered peak. Experimental procedures and limitations for measuring range-extended EXAFS past multiple K- or L-edges, and the design and operation of the spectrometer have been described previously (32Glatzel P. de Groot F.M.F. Manoilova O. Grandjean D. Weckhuysen B.M. Bergmann U. Barrea R. Phys. Rev. B. 2005; 72: 014-117Crossref Scopus (42) Google Scholar, 39Glatzel P. Bergmann U. Yano J. Visser H. Robblee J.H. Gu W.W. de Groot F.M.F. Christou G. Pecoraro V.L. Cramer S.P. Yachandra V.K. J. Am. Chem. Soc. 2004; 126: 9946-9959Crossref PubMed Scopus (161) Google Scholar). All samples were measured below 10 K in a liquid He cooled cryostat (Oxford CF1208). Data Analysis—For each EXAFS scan, the energy was calibrated using the KMnO4 pre-edge reference peak (6543.3 eV), and the intensity was normalized by I0 before averaging. Approximately 1000 scans were averaged for each orientation of PS II membranes relative to the x-ray e-vector with a custom Matlab program. Data reduction of the EXAFS spectra was done as described previously (10DeRose V.J. Mukerji I. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. J. Am. Chem. Soc. 1994; 116: 5239-5249Crossref Scopus (120) Google Scholar, 40Robblee J.H. Messinger J. Cinco R.M. McFarlane K.L. Fernandez C. Pizarro S.A. Sauer K. Yachandra V.K. J. Am. Chem. Soc. 2002; 124: 7459-7471Crossref PubMed Scopus (163) Google Scholar). Curve fitting was performed using ab initio calculated phases and amplitudes from the FEFF8 program from the University of Washington (41Rehr J.J. Albers R.C. Rev. Mod. Phys. 2000; 72: 621-654Crossref Scopus (2722) Google Scholar). These phases and amplitudes were used in the EXAFS Equation 1, which is described below and contains a sinusoidal function that gives the distance and an amplitude function that contains information about the scattering atom and the number of such neighboring atoms. χ(k)=S02∑jNjkRj2feffj(π,k,Rj)e−2σj2k2e−2Rj/λj(k)×sin(2kRj+αij(k))(Eq. 1) The neighboring atoms to the central atom(s) are divided into j shells, with all atoms with the same atomic number and distance from the central atom grouped into a single shell. Within each shell, the coordination number Nj denotes the number of neighboring atoms in shell j at a distance of Rj from the central atom, i. feffj is the ab initio amplitude function for shell j, and the Debye-Waller term e−2σj2k2 accounts for damping due to both static and thermal disorder in absorber-backscatterer distances. The mean free path term e–2Rj/λj(k) reflects losses due to inelastic scattering, where λj(k) is the electron mean free path. The oscillations in the EXAFS spectrum are reflected in the sinusoidal term sin(2kRj + αij(k)), where αij(k) is the ab initio phase function for shell j. This sinusoidal term shows the direct relation between the frequency of the EXAFS oscillations in k-space and the absorber-backscatterer distance. The EXAFS equation (Equation 1) was used to fit the experimental Fourier isolates using N, R, and σ2 as variable parameters. Fit details and evaluation of fit qualities are given in the supplemental data. The spatial resolution in EXAFS is inversely related to the spectral range. Several formulas can be found in the EXAFS literature describing the resolution limits of the method, such as ΔRΔk ≈ 1, ΔRkmax = π/2 and ΔRΔk = π/2 (40Robblee J.H. Messinger J. Cinco R.M. McFarlane K.L. Fernandez C. Pizarro S.A. Sauer K. Yachandra V.K. J. Am. Chem. Soc. 2002; 124: 7459-7471Crossref PubMed Scopus (163) Google Scholar, 42Teo B.K. EXAFS: Basic Principles and Data Analysis. Springer-Verlag, Berlin1986Crossref Google Scholar); for more details see the supplemental data (40Robblee J.H. Messinger J. Cinco R.M. McFarlane K.L. Fernandez C. Pizarro S.A. Sauer K. Yachandra V.K. J. Am. Chem. Soc. 2002; 124: 7459-7471Crossref PubMed Scopus (163) Google Scholar). For a detailed explanation of the theory of polarized EXAFS see the supplemental data. Angle θ is the angle between the x-ray e-vector and the membrane normal, and ϕ denotes the relative orientation of the manganese-backscatterer (manganese-manganese or manganese-calcium) vector of interest to the membrane normal. Manganese X-ray Absorption Spectra—X-ray absorption manganese K-edge spectra of oriented membranes in the S1 state are shown in Fig. 2. Data were collected for two orientations in which the sample normal is placed at either 15 or 75° to the direction of the e-vector of the polarized x-rays. The edge positions and post-edge shape exhibit a marked angle dependence, as reported previously for oriented PS II membranes (34Mukerji I. Andrews J.C. DeRose V.J. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1994; 33: 9712-9721Crossref PubMed Scopus (58) Google Scholar). Dichroism of the manganese K-edge spectra is even more obvious in the second-derivative spectra (Fig. 2, bottom). The powder manganese XANES spectrum created from the spectra collected from oriented membranes at two different orientations is identical to that obtained from a frozen solution sample. This result provides independent confirmation that the oriented samples are intact and not damaged by x-rays. The k3-weighted EXAFS spectra of the S1 state of PS II oriented with the membrane normal at 15 or 75° to the x-ray e-vector are shown in Fig. 3. The spectra are distinctly dichroic. The region of the photoelectron wavevector from 3.5 to 11.5 Ä–1 (denoted by the dashed line in Fig. 3), which is accessible by conventional EXAFS, agrees with results published earlier for oriented PS II membranes in the S1 state (34Mukerji I. Andrews J.C. DeRose V.J. Latimer M.J. Yachandra V.K. Sauer K. Klein M.P. Biochemistry. 1994; 33: 9712-9721Crossref PubMed Scopus (58) Google Scholar). Clear differences can be seen in the spectra between the two orientations. Fig. 4A shows the Fourier transforms of the range-extended EXAFS (k3-weighted) at 15 or 75°. The Fourier transforms exhibit well defined peaks, labeled I, II, IIIA, and IIIB, corresponding to the shells of backscatterers at different “apparent” distances, R′, from the manganese absorber. The apparent distance is shorter than the actual distance due to a phase shift induced by the interaction of the given absorber-scatterer pair with the photoelectron. For comparison, the Fourier transforms of the range-extended EXAFS spectra of both orientations, but truncated at 11.5 Ä–1, are shown in Fig. 4B. Significant improvement in spectral resolution is observed for the range-extended EXAFS data (Fig. 4A). Increased spectral resolution reveals the orientation dependence of peaks II and IIIA and IIIB. The intensity of peak II, which consists of three manganese-manganese distances at 2.7 and 2.8 Ä (see below) changes significantly between 15 and 75°, with higher intensity at 75°. Peak III shows a complex nature containing at least two peaks, IIIA and IIIB, with distances of 3.2 and 3.4 Ä, respectively. Peak IIIA is more intense at 75° but has a decreased intensity at 15°. Peak IIIB is more intense at 15° but is close to the noise level at 75°. Previous calcium EXAFS studies of native PS II (22Cinco R.M. Holman K.L.M. Robblee J.H. Yano J. Pizarro S.A. Bellacchio E
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