Secondary electron transfer reactions of the isolated Photosystem II reaction centre after reconstitution with plastoquinone-9 and diacylglycerolipids

Abstract

A Photosystem II (PS II) reaction centre preparation isolated with Triton X-100 and stabilised by transfer to dodecylmaltoside has been used in a reconstitution procedure with diacyl lipids and the naturally occurring PS II quinone, plastoquinone-9 (PQ9). As shown previously, this isolated protein complex consists of the D1, D2, b -559 α and β polypeptides, plus the psb I gene product. Before reconstitution there was only about 0.004 mol PQ9 and 0.2 mol diacyl lipid per mole chlorophyll a associated with the isolated complex. After reconstitution and precipitation by centrifugation the complex was found to have an associated lipid matrix of 1.3 mol PQ9 and 3.7 mol diacyl lipid for each mole of chlorophyll a . After reconstitution, the presence of a functional quinone in the PS II reaction centre was indicated by a strong thermoluminescence signal with a peak emission at −60°C. This was recognised as the previously characterised Z v signal on the basis of the relationship between emission and excitation temperatures and a complete quenching by ethanol. This signal was essentially unaltered by addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and was the same after reconstitution with phosphatidylcholine or a thylakoid lipid extract. Calculation of the activation free energy for the charge separation reaction which gave rise to this signal suggested that this was 0.48 ± 0.01 eV and was the same as for a smaller Z v signal detected in the isolated complex before reconstitution with PQ9. When duroquinone was used this value was not significantly different, although a much smaller signal was observed. With decylplastoquinone the thermoluminescence peak was as large as with PQ9 but the difference in shape indicated that the activation free energy was significantly higher at 0.55 ± 0.02 eV. Photosynthetic electron transfer in the reconstituted complex was assayed by reduction of 2,6-dichlorophenolindophenol (DCPIP) with diphenylcarbazide (DPC) as an artificial electron donor. A low residual rate was strongly enhanced by reconstitution with PQ9. This activity was greater when reconstitution was with a thylakoid lipid extract than with phosphatidylcholine and was inhibited by addition of DCMU ( I 50 = 0.2 mM).