The S₀ State of the Oxygen-Evolving Complex in Photosystem II Is Paramagnetic:  Detection of an EPR Multiline Signal

Abstract

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...

Stoichiometry of Proton Release from the Catalytic Center in Photosynthetic Water Oxidation

“…Only a manganese dimer was considered to be functional. The mixed valence states in S 0 and S 2 (spin ϭ 1 ⁄2) are in agreement with an EPR multiline signal in S 0 (41,42) and S 2 (43). Valence states of the manganeses have also been derived from x-ray absorption shifts at the manganese-K-edge (37-40) indicating manganese oxidations up to S 3 .…”

Focusing the view on nature's water-splitting catalyst

“…The reason for this expectation is considered to be the following: for cw X-band, the S 0 signal is usually not seen in absence of methanol, probably because of a significant broadening of the hyperfine lines under these conditions. However, with high sample concentrations we did observe a broad, featureless signal around gZ2 for the S 0 state in samples containing no methanol (Messinger et al 1997). Similarly, Thermosynechococcus elongatus preps display an S 0 EPR signal in absence of methanol (Boussac et al 1999 Mn-ENDOR spectra we derive four effective hyperfine interaction tensors, A i,iso .…”

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

“…It is notable that the distance between W3 and Ca 2ϩ /Sr 2ϩ was elongated by the Sr 2ϩ substitution, whereas the distance between W4 and Sr 2ϩ was similar to that between W4 and Ca 2ϩ (21). EPR spectroscopy has been used to characterize the manganese cluster (23)(24)(25)(26) extensively, and a typical, S 2 state multiline signal centered at g ϭ 2 has been well characterized. The multiline signal showed 19 -21 peaks in a range over ϳ180 mT width, and its properties have been studied by several advanced EPR techniques such as 55 Mn-ENDOR (27), ESEEM (28,29), and pulsed ELDOR (30).…”