Spinacia oleracea
Photosystem II enriched membrane fragments from spinach (BBY membranes) were prepared in the dark at 4°C using only green light according to a protocol, combining established procedures (Berthold et al. 1981; Volker et al. 1984; Johnson et al. 1994). 500 g fresh market spinach were carefully washed and stored overnight at 4°C in the dark to minimize the starch content in the leaves. On the following day, the spinach leaves were homogenized in a pre-cooled blender in buffer 1, containing 20 mM tricine-NaOH (pH
8.1), 0.4 M NaCl, 2 mM MgCl2, and 0.2 % BSA, freshly added at the day of the preparation.
300 mL buffer 1 were used per 100 g of spinach. The homogenate was carefully filtered through a cheese cloth into large centrifuge tubes which were kept on ice; foaming was avoided. The filtrate was centrifuged for 10 min at 6,000 x g and the supernatant was discarded. The pellet was resuspended in 80 mL buffer 1 with a
55 watercolour brush and the suspension was carefully vortexed to homogenize the solution. The solution was furthermore centrifuged at 35,000 x g for 7 min, the supernatant was discarded and the precipitate was suspended in 40 mL buffer 2, containing 20 mM MES-
NaOH (pH 6.5), 15 mM NaCl, 5 mM MgCl2, 1 mM sodium bicarbonate and 0.2 % BSA,
freshly added at the day of the preparation. The suspension was inverted about 5-6 times without foaming and centrifuged at 35,000 g for 7 min. The supernatant was discarded and the pellet was gently resuspended with a watercolour brush in 7 mL buffer 3,
containing 25 mM MES-NaOH (pH 6.5), 0.4 M sucrose, 15 mM NaCl, 5 mM MgCl2 and 1 mM
bicarbonate. The sample volume was measured in a pre-cooled graduated cylinder and the chlorophyll concentration was determined while incubating the suspension for 30 min on ice: 10 µL of the sample was added to 40 µL buffer 3. 10 µL of the diluted sample was added to 990 µL acetone (80 %). The sample was centrifuged for 2 min at 14,500 rpm in an Eppendorf centrifuge and the chlorophyll concentration was determined measuring the absorption of the sample at 663 nm (chlorophyll a) and 645 nm (chlorophyll b). The
total chlorophyll concentration was calculated according to Arnon (1949):(Arnon 1949)
[Chl] = (A663 · 8.02 + A645 · 20.2)/1000 (2.6) The chlorophyll concentration was adjusted to 3 mg/mL with buffer 3 and buffer 4 (buffer 3 with 33 w/v Triton X-100), keeping a ratio buffer 3/buffer 4 of 2/11 of the total volume and a chlorophyll to Triton X-100 ratio of 1:20 with 2/11 of the total volume being buffer 4. Foaming was avoided and the sample was incubated without stirring for 15 min when the desired concentration was achieved. The sample was centrifuged again at 35,000 x g for 15 min. The supernatant was discarded and the pellet was suspended in 40 mL buffer 3 and centrifuged at 3,000 x g for 3 min. The supernatant was centrifuged once more at 35,000 x g for 15 min. If the resulting supernatant was still green, the pellet from the previous step was suspended again in buffer 3 and centrifuged at 35,000 x g for 20 min. This was repeated until the supernatant was clear. The precipitate was suspended in a small volume of buffer 5, containing 20 mM MES-NaOH (pH 6.5), 0.4 M sucrose, 15 mM
NaCl, 5 mM MgCl2, 20 % (v/v) glycerol and 1 mM bicarbonate and the chlorophyll
concentration of the sample was determined as described above. The PSII enriched membranes were divided into aliquots, frozen with liquid nitrogen and stored at -80°C.
56 2.5.1 Preparation of Mn-depleted PSII enriched membranes
(1) Some PSII enriched membranes from spinach were depleted of the Mn4CaO5
cluster by treating them with hydroxylamine (Johnson, Krieger & Rutherford, 1995), similarly to the procedure described above for Photosystem II core complexes isolated from T. elongatus (section 2.4.1): the PSII enriched membranes were incubated for 1h on ice in the dark with [Chl] = 0.5 mg/mL in a buffer
containing 5 mM NH2OH, 400 mM NaCl and 50 mM MES-NaOH (pH 6.5). To remove
the hydroxylamine, the sample was washed at least twice in the same buffer
without NH2OH using Millipore Ultrafree-15 centrifugal filters with a 100 kDa cut-
off by centrifugation at 4,000 x g for 30 min, using the same buffer without NH2OH
and EDTA.
(2) In some samples, the Mn4CaO5 cluster was removed as well by incubating the PSII
enriched membranes in a buffer containing Tris, which was carried out as described above (section 2.4.1).
2.5.2 Oxygen evolution measurements
Oxygen evolution activity was assayed with a Clark-type oxygen electrode (Oxylab, Hansatech) at 25 °C in the presence of 0.5 mM DCBQ and 1.0 mM potassium ferricyanide,
using saturating red light (590 nm cut-off filter; 5,000 μE · m-2 · s-1). The activity in the
various preparations were about 300 - 500 μmol O2 · mg Chl a-1 · h-1 under these
conditions.
2.5.3 Removal of the bicarbonate ligand
The depletion of the bicarbonate ligand at the PSII acceptor side of PSII enriched membranes was carried out as described in Shevela et al. (2007) with some modifications:
CO2/HCO3- was removed from the buffer (400 mM sucrose, 20 mM CaCl2, 10 mM MgCl2
and 50 mM MES-NaOH, pH 6.5, BC+ buffer) by flushing with argon for 60 min (BC- buffer).
The PSII enriched membranes were depleted of CO2/HCO3- by 50-fold dilutions with the
BC- buffer and subsequent dark incubation on ice for 2 h under argon atmosphere. The
57
4,000 x g for 30 min and were washed at least twice in the BC- medium. They were stored
in the dark and on ice under argon atmosphere until they were used. If not indicated otherwise, the samples used for the spectroelectrochemical titrations were prepared freshly at the day of the experiment.
2.5.4 Treatment with sodium formate
The treatment of the PSII enriched membranes with sodium formate to replace the bicarbonate ligand was carried out according to the method described by Shevela et al.
(2007) with minor modifications: samples were treated with the BC+ buffer containing
50 mM NaHCO2 at 20 °C and pH 5.0 for 15 min. The treated samples were diluted 50-fold
in the BC- medium (pH 6.5) containing 50 mM NaHCO2. The PSII enriched membranes
were collected by centrifugation at 4,000 x g for 30 min, washed once in the same buffer and finally resuspended to the desired concentration.