Spectroscopic characterization of photoinhibited Photosystem II and kinetic resolution of the triggering of the D 1 reaction center protein for degradation

Abstract

Photoinhibition of isolated thylakoids results in inactivation of Photosystem II electron transport and proteolysis of the D 1 reaction center protein. At low, non-freezing temperatures the mechanism of inactivation for Photosystem II electron transport can be experimentally studied without interference of secondary damaging effects, since the degradation of the D 1-protein does not occur. Here we have applied electron paramagnetic resonance (EPR) spectroscopy to characterize the sequential events leading to inhibition of PS II electron transport and triggering of the D 1-protein for degradation at 2 C. Two principle kinetics of inactivation and damage were observed: (i) inactivation with a half-time of 1 to 1.5 h in case of steady-state electron transport through Photosystem II, the induction of the S 2-state multiline EPR signal, the EPR signal from Q A-Fe 2− and lowering of the F v/ F m fluorescence ratio. This is explained by over-reduction of the first quinone acceptor (Q A) leading to impairment of its function. (ii) Inhibition with a half-time of 3–4 h in case of EPR Signal II slow; inhibition of the primary charge separation reaction and release of manganese from its site in the oxygen evolving system. We were also able, for the first time, to follow the kinetics for the triggering of the D 1-protein for degradation. This triggering followed the slower kinetic phase and is likely to be the result of conformational changes in the protein induced by the highly reactive singlet oxygen. Additionally, a dark-stable cationic radical with g = 2.0031 and 10–11 G wide was progressively induced during the inhibition and was tentatively attributed to a carotenoid cation.