Electrochromic absorbance changes in relation to electron transport and energy coupling in thylakoid membranes : [Electrochrome absorptie veranderingen in relatie tot elektronentransport en energiekoppeling in thylakoid membranen]

This thesis deals mainly with the analysis and interpretation of the flash-induced electrochromic absorbance changes in isolated chloroplasts of spinach and pea plants. The amplitude and kinetics of the flash-induced absorbance changes at 518 nm (P515) are discussed in relation to the functioning of the photosynthetic electron transport and energy coupling mechanisms, which are associated with the thylakoid membrane.At least four different components can be distinguished in the flash- induced P515 signal in intact chloroplasts. Reaction 1/RC reflects the primary charge separation in the photosynthetic reaction centers. It shows a fast rise and single exponential decay kinetics. It is shown that the generally recognized "slow rising phase" in fact is composed of at least two distinguishable components related to different membrane processes. One component called reaction 1/Q is associated with the functioning of an electrogenic Q-cycle, whereas the other slow component, called reaction 2, is non-electrogenic, and is proposed to be associated with innermembrane proton domains. The two slow components can be discriminated based on their different rise and decay kinetics and the different stimulatory and inhibitory effects of DQH 2 , DBMIB and CCCP, respectively.A fourth component in the flash-induced P515 signal is called reaction 3. This component which contributes only 5-10% to the overall P515 signal is non-electrochromic and non-electrogenic. With the aid of different electron transport inhibitors and electron-donors the origin of reaction 3 is located more precisely. Under certain experimental conditions, reaction 3 exhibits a binary oscillation. Although this oscillation most probably originates from the Q B two electron gating mechanism at the reducing site of photosystem 2, it is shown that the occurence of reaction 3 in the flash-induced absorbance change is directly associated with the electron transport in photosystem 1.The kinetics of the distinguishable P515 components are studied in relation to the onset of ATP synthesis and the steady state ATP synthesis rate. The dissipation of the transmembrane electric field as monitored by reaction 1/RC is stoichiometrically related to the ATP synthesis rate. The decay of the non- electrogenic saturable component, reaction 2, is not influenced by the ATP synthesis rate. Consequently it is concluded that the decay of the overall P515 signal cannot be used as a proper measure of the ATP synthesis rate.Under the experimental conditions used, an enhanced number of flashes necessary for initiating ATP synthesis is accompanied by a lower extent of reaction 2. This finding is discussed within the frame of the earlier suggested relation between reaction 2 and the existence of innermembrane proton domains.Finally, some aspects of the use of the flash-induced P515 signal as a tool in studies of the photosynthetic electron transport and energy coupling are discussed. The P515 signal is used to monitor e.g. i) the extent of the primary charge separation, which is related to the redox state of the primary electron acceptors, ii) the functioning of an electrogenic Q-cycle and iii) the activation state of the thylakoid membranes ATPase.It is emphasized that the non-electrogenic saturable character of the reaction 2 component which is sometimes insufficiently recognized in experimental work of others, is a possible source of misinterpretations and confusion concerning the kinetics of the P515 signal