Spectral characterization in the ultraviolet region of the precursor of photosynthetically evolved oxygen in isolated trypsinized chloroplasts

Abstract

The reaction mechanism of the water-splitting enzyme system Y has been analyzed by measurements of ultraviolet absorption changes induced by a flash train in dark-adapted chloroplasts. In order to eliminate processes which gives rise to interfering absorption changes with a binary oscillation, mildly trypsizined chloroplasts were applied which are known to be disconnected from the secondary plastoquinone acceptor B, but are able to evolve oxygen, provided that K 3Fe(CN) 6 is used as electron acceptor (Renger, G. (1976) Biochim. Biophys. Acta 440, 287–300). It was found that: (a) The extent of the relaxation kinetics of the 320 nm absorption change accomplished within 5 ms oscillates with a periodicity of four in phase with the oxygen yield as a function of the number of the flash in the train. (b) The difference spectra of the difference between the initial amplitudes and those remaining after a 4.5 ms dark period, referred to as ΔA 4.5 n , exhibit pronounced bands peaking around 320 nm (positive) and 270 nm (negative). The extent oscillates in the flash train. It is at a maximum for the absorption change differences induced by the third flash, ΔA 4.5 3. (c) Taking into account the fact that the first flash of the train does not yield oxygen, whereas the oxygen production is at a maximum due to the third flash, the difference of ΔA 4.5 3 − ΔA 4.5 1 = ΔA 4.5 3,1 as a function of wavelength is interpreted as the difference spectrum of the precursor of molecular oxygen in system Y. The spectrum exhibits a band peaking around 320 nm. Accordingly, the precursor is referred to as Y-320. The data obtained are discussed in terms of a molecular model proposed for the mechanism of photosynthetic water oxidation (Renger, G. (1977) FEBS Lett. 81, 223–228).