Biochemical and Spectroscopic Characterization of Highly Stable Photosystem II Supercomplexes from Arabidopsis

Aurelie Crepin,Stefano Santabarbara,Stefano Caffarri

doi:10.1074/jbc.m116.738054

Abstract

Photosystem II (PSII) is a large membrane supercomplex involved in the first step of oxygenic photosynthesis. It is organized as a dimer, with each monomer consisting of more than 20 subunits as well as several cofactors, including chlorophyll and carotenoid pigments, lipids, and ions. The isolation of stable and homogeneous PSII supercomplexes from plants has been a hindrance for their deep structural and functional characterization. In recent years, purification of complexes with different antenna sizes was achieved with mild detergent solubilization of photosynthetic membranes and fractionation on a sucrose gradient, but these preparations were only stable in the cold for a few hours. In this work, we present an improved protocol to obtain plant PSII supercomplexes that are stable for several hours/days at a wide range of temperatures and can be concentrated without degradation. Biochemical and spectroscopic properties of the purified PSII are presented, as well as a study of the complex solubility in the presence of salts. We also tested the impact of a large panel of detergents on PSII stability and found that very few are able to maintain the integrity of PSII. Such new PSII preparation opens the possibility of performing experiments that require room temperature conditions and/or high protein concentrations, and thus it will allow more detailed investigations into the structure and molecular mechanisms that underlie plant PSII function.

Highlights

Photosystem II (PSII)2 is the first complex involved in oxygenic photosynthesis
The core complex is composed of several subunits as follows: (i) D1 and D2, which contain the reaction center P680 and the cofactors of the electron transport chain; (ii) CP47 and CP43, which act as inner antennas by coordinating most of the core chlorophyll a molecules (Chls a), and (iii) several low molecular mass subunits, whose roles for the most part are only little understood (3, 4)
We investigated the effect of increasing digitonin concentrations up to 0.4% at room temperature (Fig. 3B), and the results showed that even quite elevated digitonin concentrations have no appreciable effect on PSII integrity

Summary

Preparation and Characterization of Stable Plant PSII

The larger supercomplex observed in Arabidopsis thaliana contains a dimeric core (C2), two copies of the monomeric antenna complexes per dimer, two LHCII trimers (trimer S) strongly bound to the complex on the side of CP43 and CP26, and two more trimers, moderately bound (trimer M) in contact with CP29 and CP24 This complex is known as the C2S2M2 supercomplex (13). The protocol used for spinach PSII preparation by Wei et al (25) is the one that we previously published for obtaining homogeneous preparations of Arabidopsis PSII supercomplexes with different antenna sizes (12) Those preparations were stable only in cold conditions and for some tens of hours. CHAPSO, n-hexadecyl-␤-Dmaltoside, and Pluronic F68 were determined to be the most efficient Such a new preparation opens the possibility to investigate in much further detail the molecular mechanism underlying plant PSII activity by structural, functional, and spectroscopic techniques

Results

Discussion

Experimental Procedures