Contrary to the assertion by Preston and Critchley [(1985) FEBS Lett. 184, 318‐332] that there is no correlation between calcium and water‐soluble 23 and 17 kDa polypeptides in promoting oxygen evolution activity, it can be shown that both calcium and the polypeptides are required for activity under physiological conditions. In the absence of the two polypeptides, non‐physiological concentrations of both calcium and chloride must be present for activity; neither ion can, by itself, promote high rates of oxygen evolution.
A method is reported for the isolation of a highly resolved oxygen‐evolving photosystem II reaction center preparation. This preparation can be separated from the more complex photosystem II membranes isolated by the procedure of Berthold et al. [(1981) FEBS Lett. 134, 231‐234] by use of octylglucopyranoside at elevated ionic strengths; the oxygen‐evolving material can be collected by centrifugation at relatively low g values (40000 × g ) in yields estimated to be more than 80%. This new preparation lacks the 17 and 23 kDa extrinsic polypeptides; addition of calcium and chloride produces activities approaching 1000 μmol O 2 /h per mg chlorophyll. Although activity is maximal in the presence of 2,5‐dichloro‐β‐benzoquinone, the response of activity to ferricyanide and 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea indicates that the reducing side of photosystem II has been modified in this new oxygen‐evolving reaction center preparation.
X-ray absorption spectroscopy (XAS) has been used to characterize the structural consequences of Ca2+ replacement in the reaction center complex of the photosynthetic oxygen-evolving complex (OEC). EPR and activity measurements demonstrate that, in the absence of the 17 and 23 kDa extrinsic polypeptides, it is not necessary to use either low pH or Ca chelators to effect complete replacement of the active site Ca2+ by Sr2+, Dy3+, or La3+. The extended X-ray absorption fine structure (EXAFS) spectra for the OEC show evidence for a Mn···M interaction at ca. 3.3 Å that could arise either from Mn···Mn scattering within the Mn cluster or Mn···Ca scattering between the Mn cluster and the inorganic Ca2+ cofactor. There is no significant change in the either the amplitude or the phase of this feature when Ca2+ is replaced by Sr2+ or Dy3+, thus demonstrating that there is no EXAFS-detectable Mn···Ca contribution at ca. 3.3 Å in these samples. The only significant consequence of Ca2+ replacement is a small change in the ca. 2.7 Å Mn···Mn distance. The average Mn···Mn distance decreases 0.014 Å when Ca2+ is replaced by Sr2+ and increases 0.012 Å when Ca2+ is replaced by Dy3+. A structural model which can account both for the variation in Mn···Mn distance and for the known properties of Ca2+-substituted samples is one in which there is a hydrogen bond between a Ca2+-bound water and a Mn2(μ-O)2 unit. This scheme suggests that an important role for the Ca2+ may be to modulate the protonation state, and thus the redox potential, of the Mn cluster.
Depletion of Ca2+ and/or Cl- ions from PSII membranes blocks the electron-transfer reactions that precede O2 evolution on the oxidizing side of the enzyme. Illumination of these inhibited preparations at 273 K generates a paramagnetic species that is detectable by low-temperature (T < 20 K) EPR as a signal in the g = 2 region, 90-230 G wide, depending on the treatment that PSII has undergone. This signal has recently been assigned to YZ* in magnetic interaction with the manganese cluster in its S2 state [Gilchrist et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9545-9549]. This view, however, is not universal, owing, in part, to the fact that its spectroscopic properties depend on the preparation and the experimental conditions used for its study and, in part, to uncertainties as to the room temperature behavior of YZ* in inhibited preparations. Here, we report time-resolved and conventional EPR data showing that, at room temperature and at 273 K, YZ* can be accumulated in its 20 G form in high yields in both Ca2+-depleted and acetate-inhibited preparations, and that the kinetics of its decay match the decay kinetics of the low-temperature signal generated in corresponding samples. The properties of the YZ* signal, however, are shown to depend on the polypeptide content, the temperature, and the electron donors and acceptors present in the sample under examination. Our results support assignment of the EPR signal in inhibited preparations to S2 YZ* and demonstrate a protective role of the 17 and 23 kDa extrinsic polypeptides for the manganese cluster against externally added reductants.
