Reconstitution of the Water-Oxidizing Complex in Manganese-Depleted Photosystem II Complexes by Using Synthetic Binuclear Manganese Complexes.

Abstract

The efficiency of synthetic binuclear manganese complexes in reconstituting PS II electron flow and oxygen-evolution capacity was analyzed in PS II enriched preparations deprived of their manganese and of the extrinsic regulatory subunits. Measurements of the variable fluorescence induced by actinic illumination with continuous light led to the following results: (a) the synthetic binuclear complexes are more efficient than MnCl2 in establishing a PS II electron flow; (b) an almost complete restoration is achieved at concentrations of these complexes that correspond with an overall stoichiometry of two manganese per PS II; and (c) the electron flow restored by the binuclear manganese complexes closely resembles that of normal O2-evolving PS II preparations in its resistance to addition of 50 microM EDTA, while that supported by MnCl2 is practically completely suppressed at the same chelator concentration. The rate of O2 evolution was used as a measure of the capability to function as manganese source in reconstitution of the oxygen evolution capacity. It was found that (i) as in the case of PS II electron transport, the synthetic binuclear manganese complexes are significantly more efficient than MnCl2; (ii) with respect to the manganese concentration, the maximum effect is achieved with a mu-oxo bridged binuclear Mn(III) complex (symbolized by M-3) at concentrations corresponding to four manganese per PS II; and (iii) at all concentrations of binuclear manganese complex M-3 a significantly higher restoration of the O2 evolution rate is achieved if the reconstitution assay contains in addition the extrinsic regulatory 33 kDa protein (PS II-O protein).(ABSTRACT TRUNCATED AT 250 WORDS)

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem II

“…3 shows the double reciprocal plots of this dependence at pH 5. (20,21). In A, 0.4 M MnCl2 (2 Mn͞RC) was added.…”

“…The BBY [subchloroplast preparation isolated by the method worked out by Berthold, Babcock, and Yocum (18)] PSII membrane fragments were prepared as described (18,19). A complete (Ͼ95%) removal of Mn from the membrane fragments was carried out using 1 M Tris⅐HCl (pH 8.0) plus 0.5 M MgCl 2 (17) or N,N,NЈ,NЈ-tetramethylethylenediamine (TEMED) (20,21) treatments. The preparations were stored in liquid nitrogen or at Ϫ80ЊC at a Chl concentration of 10 mg͞ml after the addition of 10% glycerol or 0.4 M sucrose to the medium.…”

Photoinhibition of Photosystem II from Higher Plants

“…It seems, therefore, that Cu(II) inhibitory and TEMED treatment effects are the consequence of different molecular mechanisms. Since TEMED treatment removes the extrinsic proteins and the manganese cluster of the PSII donor side (35), it can be suggested that the Cu(II)-inhibitory site, in these experimental conditions, is not located on the donor side of PSII. In addition, the presence of Cu(II) did not modify the D1 protein degradation in the non-oxygenic (i.e.…”

“…This sample exhibited oxygen evolution rates of 560 mol of O 2 ⅐mg of Chl Ϫ1 ⅐h Ϫ1 in the presence of 0.5 mM 2,6-dichlorobenzoquinone as artificial electron acceptor. Non-oxygenic PSII membranes were prepared by incubation of the intact PSII membranes (0.2 mg of Chl⅐ml Ϫ1 ) in a suspension containing 20 mM TEMED, 0.5 M MgCl 2 , and 20 mM Mes-NaOH, pH 6.5 (35). After centrifugation at 20,000 ϫ g the pellet was washed twice in a buffer solution of 35 mM NaCl and 20 mM Tris-HCl, pH 8.0.…”

“…After centrifugation at 20,000 ϫ g the pellet was washed twice in a buffer solution of 35 mM NaCl and 20 mM Tris-HCl, pH 8.0. This procedure extracts manganese and the three extrinsic proteins with molecular masses of 33, 24, and 18 kDa (35). The oxygen evolution capacity was completely eliminated in these manganese-depleted preparations.…”