Dorianna Sandonà

CHLOROPHYLL BINDING TO MONOMERIC LIGHT-HARVESTING COMPLEX : A MUTATION ANALYSIS OF CHROMOPHORE-BINDING RESIDUES

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 refoldedin 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 Qy 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.

Higher plants light harvesting proteins. Structure and function as revealed by mutation analysis of either protein or chromophore moieties

Mutation analysis of higher plants light harvesting proteins has been prevented for a long time by the lack of a suitable expression system providing chromophores essential for the folding of these membrane-intrinsic pigment-protein complexes. Early work on in vitro reconstitution of the major light harvesting complex of photosystem II (LHCII) indicated an alternative way to mutation analysis of these proteins. A new procedure for in vitro refolding of the four light harvesting complexes of photosystem II, namely CP24, CP29, CP26 and LHCII yields recombinant pigment-proteins indistinguishable from the native proteins isolated from leaves. This method allows both the performing of single point mutations on protein sequence and the exchange of the chromophores bound to the protein scaffold. We review here recent results obtained by this method on the pigment-binding properties, on the chlorophyll-binding residues, on the identification of proton-binding sites and on the role of xanthophylls in the regulation of light harvesting function.

A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29

Energy‐dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein‐modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light‐harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild‐type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.

The photosystem II subunit CP29 can be phosphorylated in both C3 and C4 plants as suggested by sequence analysis

The CP29 subunit of Photosystem II is reversibly phosphorylated in Zea mays upon exposure to high light in the cold (Bergantino et al., J Biol Chem 270 (1995) 8474–8481). This phenomenon was previously proposed to be restricted to C4 plants. We present the complete sequence of the CP29 protein, deduced from a maize Lhcb4 cDNA clone, and its comparison with the previously known Lhcb4 sequences of two C3 plants: Hordeum vulgare and Arabidopsis thaliana. Despite the relatively low degree of homology in their amino-terminal region, i.e. the part of the molecule which is phosphorylated in maize, the three polypeptides conserve consensus sequences for the site of phosphorylation. We proved by immunoblotting and 33P-labelling that the same post-translational modification occurs in barley. Being thus common to C3 and C4 plant species, the phosphorylation of this minor antenna complex of Photosystem II appears now as a widespread phenomenon, possibly part of the phosphorylation cascade which signals the redox status of the plastoquinone to the nuclear transcription apparatus. Arabidopsis plants do not show phosphorylation of CP29 in the same conditions, but other low-molecular-weight phosphoproteins, whose role need to be elucidated, become evident.

A structural investigation of the central chlorophyll a binding sites in the minor photosystem II antenna protein, Lhcb4

Mutant proteins from light-harvesting complexes of higher plants may be obtained by expressing modified apoproteins in Escherichia coli, and reconstituting them in the presence of chlorophyll and carotenoid cofactors. This method has allowed, in particular, the engineering of mutant LHCs in which each of the residues coordinating the central Mg atoms of the chlorophylls was replaced by noncoordinating amino acids [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10056-10061]. The availability of these mutants is of particular importance for determining the precise position of absorption bands for the different chlorophyll molecules, as well as the sequence of energy transfer events that occur within LHC complexes, provided that the structural impact of each mutation is precisely evaluated. Using resonance Raman spectroscopy, we have characterized the pigment-protein interactions in the minor photosystem II antenna protein, Lhcb4 (CP29), in which each of three of the four central chlorophyll a molecules has been removed by such mutations. By comparing the spectra of these mutants with those of the wild-type protein, the state of interaction of the carbonyl group, the coordination state of the central magnesium ion, and the dielectric constant (polarity) of the immediate environment in the binding pocket of the chlorophyll a molecule were defined for each cofactor binding site. In addition, the structural impact of the absence of one chlorophyll a molecule and the quality of protein folding were evaluated for each of these mutated polypeptides.

Mutation analysis of either protein or chromophore moieties in Higher Plants Light Harvesting Proteins

In higher plants chloroplasts, chlorophyll and carotenoid molecules are non-covalently bound to specific transmembrane proteins. This polypeptide family, called Lhc includes at least 10 members in higher plants (1). Our knowledge of Lhc protein structure arises from the resolution of higher plants LHCII complex at near-atomic resolution (2) and from the conclusion that this structure provides a general model for the overall folding of all the Lhc proteins since they have substantial regions of sequence conservation (2,3) noticeably in the membrane spanning domains (4). The pigment-binding characteristics of Lhcb gene products are summarised in Table I. Since LHCII structure has been experimentally determined, this model system can be used as a guideline for mutation analysis on other members of this family. A number of drawbacks, in fact, make LHCII itself unsuitable, namely: i) it is an heterogeneous protein made by the products of many high homologous genes (5) thus making the comparison of a recombinant protein with its native counterpart impossible; ii) it is a hetero-trimeric protein in which protein-protein interactions are as important as the intra-subunit features in determining the biochemical and spectroscopical characteristics of the system thus making it difficult to identify the primary effect of point mutations; iii) it has a variable carotenoid content depending on growth conditions and occupancy of two sites whose location in the protein has not been resolved by structural studies; iv) four chlorophyll binding ligands have not been identified making them unaccessible to mutation analysis. For the above reasons, we have instead chosen Lhcb4 (CP29) since it is homogeneous, monomeric, has only two xanthophyll binding sites and 8 chlorophyll binding sites for 7 of which the ligand can be easily identified by homology with LHCII. The CP29 apoprotein is 257 amino acids long and each polypeptide binds 6 chl a 2 chl b and 2 xanthophylls (lutein, violaxanthin and neoxanthin in non stoichiometric amounts thus implying that the two sites can accomodate different carotenoids with similar structure) (2,6,7).