The oxygen-evolving complex of Photosystem II in plants and cyanobacteria catalyzes the oxidation of two water molecules to one molecule of dioxygen. A tetranuclear Mn complex is believed to cycle through five intermediate states (S 0 -S 4 ) to couple the four-electron oxidation of water with the one-electron photochemistry occurring at the Photosystem II reaction center. We have used X-ray absorption spectroscopy to study the local structure of the Mn complex and have proposed a model for it, based on studies of the Mn K-edges and the extended X-ray absorption fine structure of the S 1 and S 2 states. The proposed model consists of two di-μ-oxo-bridged binuclear Mn units with Mn-Mn distances of ~2.7 Å that are linked to each other by a mono-μ-oxo bridge with a Mn-Mn separation of ~3.3 Å. The Mn-Mn distances are invariant in the native S 1 and S 2 states. This report describes the application of X-ray absorption spectroscopy to S 3 samples created under physiological conditions with saturating flash illumination. Significant changes are observed in the Mn-Mn distances in the S 3 state compared to the S 1 and the S 2 states. The two 2.7 Å Mn-Mn distances that characterize the di-μ-oxo centers in the S 1 and S 2 states are lengthened to ~2.8 and 3.0 Å in the S 3 state, respectively. The 3.3 Å Mn-Mn and Mn-Ca distances also increase by 0.04-0.2 Å. These changes in Mn-Mn distances are interpreted as consequences of the onset of substrate/water oxidation in the S 3 state. Mn-centered oxidation is evident during the S 0 →S 1 and S 1 →S 2 transitions. We propose that the changes in Mn-Mn distances during the S 2 →S 3 transition are the result of ligand or water oxidation, leading to the Supporting Information Available: E-space S 3 state EXAFS spectrum; the data in k-space and the background that was removed to reduce the low-frequency contributions that show up as peaks at <1 Å in the Fourier transform; and the Fourier isolate of the k-space S 3 spectrum, shown overplotted on the S 3 EXAFS spectrum (PDF). This material is available free of charge via the Internet at http:// pubs.acs.org. NIH Public Access

The photosynthetic oxidation of water to molecular oxygen is energetically driven by lightinduced charge separations in the reaction center of photosystem II (PS II). The reaction is catalyzed by a tetranuclear manganese cluster contained in the oxygen-evolving complex (OEC). The OEC cycles through five different redox states termed S 0 to S 4 , with S 1 being the dark-stable state. Oxygen is released during the S 4 → S 0 transition. 1 The removal of one electron from the OEC on each S state transition leads to the idea that alternate S states should be paramagnetic because of their odd-electron number. The multiline EPR signal, which is the hallmark of the S 2 state, establishes the odd-electron character of the Mn cluster in S 2 . 2 The S 1 state, one-electron reduced from S 2 , is paramagnetic but of even electron number, and a non-Kramers EPR signal is observed in parallel-polarized EPR. 3 Because the S 0 state is reduced by one further electron, it is expected to be an odd-electron or Kramers state observable with conventional EPR. Hence, it was somewhat surprising that no EPR signal had been reported for this state. This problem was recently resolved by Messinger et al. who observed a new EPR multiline signal in an S 0 * state, an S 0 -like state produced by reduction of the S 1 state by hydroxylamine or hydrazine. 4 The essential ingredient was the addition of 1.5% methanol. We now report the observation of this EPR signal in a physiological S 0 state produced by three-flash illumination of dark-adapted PS II membranes. This EPR signal is sufficiently similar to that produced by NH 2 OH treatment so that, from the perspective of EPR, one need no longer distinguish the states prepared by the two methods. Furthermore, we describe a broad EPR signal for the S 0 state in absence of methanol.Dark-adapted spinach PS II membranes 5 were enriched in S 0 by the following flash procedure: aliquots were illuminated at a chlorophyll (Chl) concentration of 1 mg/mL in icecold pH 6.5 buffer (5 mM CaCl 2 , 5 mM MgCl 2 , 15 mM NaCl, 50 mM MES, 400 mM sucrose) with one preflash (Xe flash lamp, 13 μs FWHM, 5 J per pulse; pathlength ~ 2 mm), further dark-adapted on ice for 90-120 min, and illuminated with three Xe flashes (0.5 Hz). Before centrifugation (30 min, 40 000 × g, 4 °C) 1.5% methanol (v/v), 20 μM phenyl-p-© 1997 American Chemical Society † Structural Biology Division. ‡ Department of Chemistry.Supporting Information Available: Simulated EPR spectra (9 pages). See any current masthead page for ordering and Internet access instructions. Figure 1A shows an EPR difference spectrum from the S 0 sample (minus S 1 ) prepared with FCCP, PPBQ, and methanol. A multiline signal clearly different from the well-known S 2 multiline signal ( Figure 1B, same additions) is observed. Most of the peaks are out of phase between the two signals (see dashed lines in Figure 1). The average splitting of the hyperfine lines is very similar, about 85-90 G, but the values for the S 0 signal are more variable (70-110 G) than those for the S...

Hydrogen bond networks in protonated acetone/water clusters are stabilized by H3O+(Me2CO)2 centers, and the stabilizaton increases with further acetone content. For example, proton transfer from neat water (H2O)6H+ clusters to form mixed (Me2CO)3(H2O)3H+ clusters is exothermic by 80 kJ/mol (19 kcal/mol), due to strong hydrogen bonding of the carbonyl groups; in a series of mixed clusters B3(H2O)3H+, the stability of the hydrogen bond network correlates with the proton affinities PA(B). In diketone models of adjacent peptide links, the proton is stabilized by internal hydrogen bonds between the carbonyl groups. The internal bonds can be significant, for example, 31 kJ/mol (7 kcal/mol) in (MeCOCH2CH2COMe)H+, but proton transfer through the internal bond has a high barrier. However, water molecules can bridge between the CO groups. In these bridges, the proton remains on an H3O+ center, in both acetone/water and diketone/water systems. With a further H2O molecule, the diketone/water cluster (MeCOCH2CH2COMe)(H2O)2H+ and diamide/water clusters form two-water H3O+···H2O bridges, which allow proton transfer between the CO groups with a small barrier of <12 kJ/mol (<3 kcal/mol). The cluster models suggest several roles for hydrogen bonds in proton transport through membranes. (1) Ionic hydrogen bonds involving polar amide groups stabilize ions by up to 135 kJ/mol (32 kcal/mol) in clusters and can similarly stabilize ions in membrane water chains and enzyme centers. (2) The proton can remain on an H3O+ center and, therefore, remain delocalized and mobile in water chains, despite the stronger basicities of the surrounding amide groups. This effect results from electrostatic balancing of opposing peptide amide dipoles. (3) In the water chains, H3O+···H2O bridges between peptide amide groups can provide low-energy pathways for proton transport.