Biochemical Properties of the PsbS Subunit of Photosystem II Either Purified from Chloroplast or Recombinant

Abstract

The biochemical properties of PsbS protein, a nuclearencoded Photosystem II subunit involved in the high energy quenching of chlorophyll fluorescence, have been studied using preparations purified from chloroplasts or obtained by overexpression in bacteria. Despite the homology with chlorophyll a/b/xanthophyllbinding proteins of the Lhc family, native PsbS protein does not show any detectable ability to bind chlorophylls or carotenoids in conditions in which Lhc proteins maintain full pigment binding. The recombinant protein, when refolded in vitro in the presence of purified pigments, neither binds chlorophylls nor xanthophylls, differently from the homologous proteins LHCII, CP26, and CP29 that refold into stable pigment-binding complexes. Thus, it is concluded that if PsbS is a pigment-binding protein in vivo, the binding mechanism must be different from that present in other Lhc proteins. Primary sequence analysis provides evidence for homology of PsbS helices I and III with the central 2-fold symmetric core of chlorophyll a/b-binding proteins. Moreover, a structural homology owed to the presence of acidic residues in each of the two lumen-exposed loops is found with the dicyclohexylcarbodiimide/Ca 2؉ -binding domain of CP29. Consistently, both native and recombinant PsbS proteins showed [ 14 C]dicyclohexylcarbodiimide binding, thus supporting a functional basis for its homology with CP29 on the lumen-exposed loops. This domain is suggested to be involved in sensing low luminal pH. Photosystem II (PSII)1 of higher plants is a multisubunit membrane complex composed of many polypeptides that are encoded by the chloroplast or nuclear genes. Chloroplast gene products are located in the core complex where electron transport reactions are catalyzed, whereas the surrounding lightharvesting system is composed of nuclear-encoded chlorophyll a/b/xanthophyll proteins belonging to the Lhc family (1). Lhc proteins of PSII include Lhcb1-3 gene products that form heterotrimeric complexes (LHCII) located peripherally in the PSII⅐LHCII supercomplex and Lhcb4 -6 proteins, which form a layer of monomeric subunits located between the core complex and trimeric LHCII. Besides the role of harvesting light and transferring excitation energy to the PSII reaction center (RC), Lhc proteins are involved in regulative mechanisms aimed at both the optimization of excitation energy distribution between PSI and PSII and the protection of the PSII RC from photoinhibition when absorbed light exceeds the electron transport capacity of the chloroplast. The former mechanisms acting at moderate to low light intensity include the reversible phosphorylation of LHCII, which leads to the detachment of phospho-LHCII from PSII and its migration to stroma membranes where it transfers energy to PSI (2). The mechanisms of protection from photoinhibition are elicited at high light intensity and include the reversible phosphorylation of Lhcb4 (CP29) (3) and the xanthophyll cycle-dependent non-photochemical energy quenching (NPQ). In excess light conditions...

Fluorescence as a Tool to Understand Changes in Photosynthetic Electron Flow Regulation

“…In higher plants the 'special' polypeptide which undergoes protonation is PsbS . The function of PsbS is essentially to sense the lumen pH, that is linked to several H + -binding amino acid residues present on the luminal loops of this protein (Dominici et al 2002;Li et al 2002). In green microalgae (Peers, G and Niyogi, KK, personal communication, 2008) and diatoms (Zhu and Green 2008), the Li-818 proteins which are up-regulated under high light, could play a similar role as PsbS in qE.…”

A Mechanism of Nonphotochemical Energy Dissipation, Independent from PsbS, Revealed by a Conformational Change in the Antenna Protein CP26

“…The second type of quenching, known as qE, is pH dependent and requires protonation of lumenal exposed acidic residues in the PsbS subunit (Li et al, 2002b), which has DCCD binding sites (Dominici et al, 2002). Both types of quenching have been proposed to imply conformational changes in LHC proteins (Gilmore and Ball, 2000;Horton et al, 2000).…”

“…The interchange of these carotenoids via the intermediate antheraxanthin (Anthera), reversible in the dark by zeaxanthin epoxidase, is called xanthophyll cycle. Low lumenal pH also results in protonation of specific light-harvesting complexes (LHCs) (Dominici et al, 2002;Li et al, 2002a;Li et al, 2002b;Pesaresi et al, 1997;Walters et al, 1996), which, synergistically with zeaxanthin binding, leads to a conformational change necessary for NPQ (Moya et al, 2001). The PSII subunit S (PsbS) was found to be essential for NPQ (Li et al, 2000) and may trigger the quenching in a series of steps involving protonation and, possibly, zeaxanthin binding (Li et al, 2002b;Holt et al, 2005).…”

The Functional Significance of the Monomeric and Trimeric States of the Photosystem II Light Harvesting Complexes

“…In Chlamydomonas, elimination of one of the major LHCIItype complexes had a significant effect on qE, although in plants the lack of Lhcb1 and Lhcb2 only reduced qE capacity by about 20% (23). Mutants lacking the Lhc-related protein PsbS almost completely lack qE (24), although the exact role of PsbS is unclear, partially due to uncertainties of its pigment binding capabilities (25)(26)(27)(28)(29)(30). It has been suggested that PsbS may be a regulatory subunit in qE rather than being the site of quenching per se (31), providing binding sites for protons (32) and zeaxanthin (33).…”