Chlorophyll Binding to Monomeric Light-harvesting Complex

Abstract

The chromophore binding properties of the higher plant light-harvesting complex II have been studied by site-directed mutagenesis of pigment-binding residues. Mutant apoproteins were overexpressed in Escherichia coli and then refolded in vitro with purified chromophores to yield holoproteins selectively affected in chlorophyll-binding sites. Biochemical and spectroscopic characterization showed a specific loss of pigments and absorption spectral forms for each mutant, thus allowing identification of the chromophores bound to most of the binding sites. On these bases a map for the occupancy of individual sites by chlorophyll a and chlorophyll b is proposed. In some cases a single mutation led to the loss of more than one chromophore indicating that four chlorophylls and one xanthophyll could be bound by pigment-pigment interactions. Differential absorption spectroscopy allowed identification of the Q y transition energy level for each chlorophyll within the complex. It is shown that not only site selectivity is largely conserved between light-harvesting complex II and CP29 but also the distribution of absorption forms among different protein domains, suggesting conservation of energy transfer pathways within the protein and outward to neighbor subunits of the photosystem.In green plants light energy for photosynthesis is collected by an antenna system, made of many homologous proteins belonging to the Lhc multigene family (1). These pigment proteins are organized around photosynthetic reaction centers to form supramolecular complexes embedded into the thylakoid membranes. Lhc proteins bind about 70% of the pigments involved in plant photosynthesis. Understanding of energy transfer processes in the antenna and reaction centers requires recognition of the topological organization of subunits (2-4) and knowledge of three major parameters, namely: (i) the distances between chromophores; (ii) the mutual orientation of dipole transition moments; and (iii) the absorption/fluorescence energy levels. Although the elucidation of LHCII structure at 3.4 Å resolution (5) has allowed localization of chlorophyll-binding sites and of their relative distances, identification of transition dipole orientation and energy levels are precluded by insufficient resolution of the structure so far obtained or are not accessible by structural methods. Among Lhc proteins the most abundant is LHCII, which can be isolated as an heterotrimeric complex of the Lhcb1-3 gene products (6). LHCII binds 7 Chl 1 a, 5 Chl b, and three xanthophyll molecules/mol of polypeptide (1.6 -1.8 mol of lutein, 0.2-0.4 mol of violaxanthin, and 1.0 mol of neoxanthin) and is the best characterized Lhc polypeptide. (5, 7-10). Knowledge of the energy transfer factors for this protein would be a major step toward elucidation of light harvesting function. In this study we have used mutation analysis with the aim of the identification and characterization of the chromophores bound to each site; a series of mutant apoproteins was constructed by overexpression in bacteria ...

The Nature of a Chlorophyll Ligand in Lhca Proteins Determines the Far Red Fluorescence Emission Typical of Photosystem I

“…Considering that the 675-nm band is the only negative contribution observed in the spectra, it can be concluded that the monomers are isoenergetics. Similar values for the absorption of monomeric Chl A5 were found in LHCII and CP29 (21,22), thus supporting the present conclusion. We have to highlight the fact that the bandwidth of the red-most transition is very large, as is expected in the case of a Chl dimer because of strong electronphonon coupling, whereas the absorption at 675 nm has the FWHM typical of a Chl monomer.…”

“…It has to be considered that the His versus Asn substitution appears to be a usual tool for modulation of Chl-Chl interactions in Lhc proteins; Chl A2, when coordinated by Asn in Lhcb1, establishes excitonic interactions with neighbor pigments, namely Chl B2 and Chl A4. This interaction is absent in Lhcb4 (CP29) in which His is the ligand for Chl A2 (21,22) and again present in the Asn-bearing Lhcb5 (CP26), whose overall biochemical and spectral characteristics are very close to those of CP29 (19).…”

“…In reaction centers, the presence of dimers of the Chl molecule confers the "special" nature of a primary donor. In antenna proteins, energy levels of each individual chlorophyll chromophore are modulated by interactions with amino acid groups and carotenoid molecules to optimize both energy transfer and photoprotection (21)(22)(23). In Photosystem II, the protein environment also provides modulation of chlorophyll fluorescence lifetime and, thus, of the efficiency of energy transfer to P680 by undergoing conformational changes induced by the binding of violaxanthin versus zeaxanthin to an allosteric site called L2 (24).…”

“…WT and mutant apoproteins were isolated from the SG13009 strain of Escherichia coli transformed with constructs following a protocol described previously (17,(21)(22)(23). Reconstitution and purification of protein-pigment complexes were performed as described (11,12).…”

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

“…Two positive bands at 662 and 686 nm accompany the negative band at 675 nm, and the asymmetry of the positive band at 686 nm suggests that the bands at 662 and 686 nm arise from a blue shift and a broadening of the 675 nm band. The 675 nm absorption in the trimeric LHCII is most likely from either Chl a 4 or Chl a 5 (52), which appear to be in close proximity to Lut2 (53). Thus, these differences in absorption spectrum between LHCII trimers and monomers can tentatively be attributed to a change in the Lut2/ Chl a 4 /Chl a 5 domain occurring upon monomerization.…”