Effect of partial removal and readdition of a 23 kilodalton protein on oxygen yield and flash-induced absorbance changes at 320 nm of inside-out thylakoids

Abstract

Washing inside-out spinach thylakoids with 250 mM NaCl at pH 7.4 causes up to 75% inhibition of oxygen evolution which has been shown by reconstitution experiments to be due mainly to the removal of a 23 kDa protein (Åkerlund, H.E., Jansson, C. and Andersson, B. (1982) Biochim. Biophys. Acta 681, 1–10). Here we have found the same degree of inhibition and reconstitution of oxygen evolution measured either in flash light or in continuous light at different light intensities, which suggests that the salt-washing causes a complete block of electron transport in certain chains rather than a decreased rate in all chains. Flash-induced absorbance changes at 320 nm, reflecting mainly the electron transfer at the Photosystem-II acceptor side, from the primary to the secondary plastoquinone in their semiquinone form (PQ − APQ − B → PQ APQ 2− B) and to minor extent the donor side of Photosystem II (Renger, G. and Weiss, W. (1983) Biochim. Biophys. Acta 722, 1–11), were measured under repetitive excitation conditions. Salt-washing the inside-out thylakoids caused a change in decay rate from 500–600 μs down to 200 μs, interpreted as an increased proportion of recombination between PQ − A and chlorophyll- a + II. Addition of the 23 kDa protein restored the kinetics up to some 400 μs. Measurements made on dark-adapted material gave evidence that salt-washing also affected S-state turnover. We conclude that the 23 kDa protein functions on the water-splitting side of Photosystem II and is indispensible for the photosynthetic water oxidation.