Fluorescence decline as a function of redox potential and actinic light intensity in spinach thylakoids

Abstract

Excitation of isolated thylakoids with sufficiently strong actinic light increases the fluorescence quantum yield up to a maximum level, F max, followed by a slower decline under certain experimental conditions. In this study the latter effect was analyzed as a function of the ambient redox potential and the actinic light intensity. Two different types of fluorescence decrease were found. (a) In the presence of specific quinones widely used as redox mediators a fast and comparatively small decrease (30% of F max), referred to as ΔF SQ, was observed at moderate redox potentials (−300 < E m < + 200 mV). ΔF SQ disappears at positive values with E m, 7.5 = + 110 mV, whereas the decrease at negative redox potential depends on the midpoint potential of the quinone. (b) A more pronounced fluorescence decline was observed at redox potentials below −300 mV, which comprises 65–70% of the maximum fluorescence. The full expression of this effect, referred to as ΔF max LP, requires markedly higher actinic light intensities than ΔF max SQ. The extent of ΔF max LP as a function of the redox potential is dependent on the presence of redox mediators. In their absence the full expression of ΔF max LP can be only observed below −400 mV. Based on the hypothesis of Pheo − photoaccumulation being responsible for the fluorescence decline at low redox potentials (Klimov, V.V., Klevanik, A.V. and Shuvalov, V.A. (1977) FEBS Lett. 82, 182–186), a reaction scheme is presented that qualitatively describes the time course of ΔF LP at different actinic light intensities and redox potentials. Based on this analysis, the rate of Pheo ⨪ reoxidation is inferred to be limited by the reaction center apoprotein acting as a barrier to redox equilibration. The implications for the interpretations of redox titration curves are briefly discussed.