Photobleaching of chlorophyll in light-harvesting complex II
Light-induced photobleaching of chlorophylls in the major trimeric light-harvesting complex II (LHCII) from Pisum sativum is investigated in different molecular environments – protein aggregates, embedded in detergent micelles (beta-dodecyl maltoside, DDM) or in reconstituted membranes with thylakoid membrane lipids. Absorption, circular dichroism, and fluorescence spectra are recorded before and at different intervals after illumination with strong white light (2000 umol photons/m2/s). EPR spectra are recorded in the presence of spin trap (TEMPD) and spin label (5-SASL) to detect reactive oxygen species. The rate of photobleaching depends on the molecular environment of the complex. Photobleaching is exacerbated in reconstituted lipid membranes. EPR spectroscopy using spin labels confirms the increased light-induced generation of singlet oxygen in the membranes. The increased susceptibility to photodamage of LHCII in lipid membranes is potentially of great significance considering that this is the native environment for the majority of photosynthetic pigment–protein complexes.
Steps to reproduce
Trimers, higher-order aggregates and reconstituted membranes of light-harvesting complex II were prepared as described by Akhtar et al. (J. Biol. Chem., 2015, 290, 4877-4886). The samples were diluted to absorbance 0.4 at 675 nm and illuminated with white light from a Schott KL-2500 LED light source in a cuvette with 1 cm pathlength at incident light intensity of 3000 umol photons/m2/s. Absorption and CD spectra were recorded with a Jasco J-815 spectrometer. Fluorescence spectra were recorded with a Jasco FP-8500 fluorometer. X-band EPR spectra were recorded with a 9 MHz microwave frequency and 1/3 G modulation using a Brucker ELEXSYS E580 spectrometer.