Analysis of Light-Induced Absorbance Changes in the Near-Infrared Spectral Region I. Characterization of Various Components in Isolated Chloroplasts

Abstract

Light-induced absorbance changes in the near-infrared (NIR ) spectral region (700-950 nm) are analyzed in isolated spinach chloroplasts using pulsed light-emitting diodes as modulated measuring light sources. With chloroplasts under coupled conditions the light-induced signal is dominated by a relatively slow scattering change displaying a flat difference spectrum. More specific changes can be distinguished by faster induction and relaxation kinetics and in the presence of valinomycin/nigericin which prevents scattering changes. Besides the dominant P700 change, with a narrow bleaching band around 703 nm and a broad absorbance increase between 750 and 850 nm (peak at 815 nm) the following absorbance changes were identified in the NIR: 1) An absorbance increase caused by plastocyanin (PC) oxidation, with a relatively flat spectrum. 2) An absorbance decrease gaining amplitude towards longer wavelengths, which reflects reduction of a low potential acceptor of PSI different from ferredoxin. 3) A field indicating absorbance decrease peaking around 730 nm, the properties of which correspond to those of P515. 4) An uncoupler insensitive absorbance decrease stimulated by dark adaptation and anaerobic conditions, the difference spectrum of which resembles that of ferredoxin reduction. The relative contributions of P700 and PC to the overall oxidized-reduced difference spectrum are determined by redox titration. At 706 nm, 815 nm and 950 nm the P 700/PC is - 119/ 19, 67/37 and 31/69, respectively. From these ratios and the molar extinction coefficients a molar P 700/PC stoichiometry of 1 /3 is determined.