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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.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Förster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

The chromophore-binding properties of the higher plant light-harvesting protein CP29 have been studied by using site-directed mutagenesis of pigment-binding residues. Overexpression of the apoproteins in bacteria was followed by reconstitution in vitro with purified pigments, thus obtaining a family of mutant CP29 proteins lacking individual chromophore-binding sites. Biochemical characterization allowed identification of the eight porphyrins and two xanthophyll-binding sites. It is shown that the four porphyrin-binding sites (A1, A2, A4, and A5) situated in the central, twofold-symmetrical domain of the protein are selective for Chl-a, whereas the four peripheral sites (A3, B3, B5, and B6) have mixed Chl-a-Chl-b specificity. Within a site, porphyrin coordination by glutamine increases affinity for Chl-b as compared with glutamate. Xanthophyll site L1 is occupied by lutein, whereas site L2 can bind violaxanthin or neoxanthin. The protein is relatively stable when site L2 site is empty, suggesting that xanthophylls can be exchanged during operation of xanthophyll cycle-dependent photoprotection mechanism. Differential absorption spectroscopy allowed determination of transition energy levels for individual chromophores, thus opening the way to calculation of energy-transfer rates between Chl in higher plant antenna proteins.

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.

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.

The minor photosystem II antenna complex CP29(Lhcb-4) has been reconstituted in vitro with the Lhcb-4 apoprotein, overexpressed in Escherichia coli, and the native pigments. Modulation of the pigment composition during reconstitution yields binding products with markedly different chlorophyll a/b binding ratios even though the total number of bound chlorophylls (a plus b) remains constant at eight. A thermodynamic analysis of steady state absorption and fluorescence spectra demonstrates that all chlorophylls are energetically coupled, while the kinetics of chlorophyll photooxidation indicate that triplet chlorophyll-carotenoid coupling is also conserved during pigment binding in vitro. The influence of the chlorophyll a/b binding ratio on the absorption spectra measured at 72 and 300 K is analyzed for the Qy absorption region. Increased chlorophyll b binding leads to large increases in absorption in the 640-660 nm region, while absorption in the 675-690 nm interval decreases markedly. These changes are analyzed in terms of a Gaussian decomposition description in which the eight subbands display a temperature-dependent broadening in agreement with the weak electron-phonon coupling demonstrated for other antenna chlorophyll spectral forms. In this way, we demonstrate that increased chlorophyll b binding leads to increased absorption intensity associated with the subbands at 640, 648, 655, and 660 nm and decreased intensity for the long wavelength subbands at 678 and 684 nm. The wavelength position of all subbands is unchanged. The above data are interpreted to indicate that CP29 has eight chlorophyll binding sites, many or all of which can be occupied by either chlorophyll a or chlorophyll b according to the conditions in which pigment binding occurs. Chlorophyll b absorption is primarily associated with four subbands located at 640, 648, 655, and 660 nm. The invariance of the wavelength position of the absorption bands in recombinant products with different chlorophyll a/b binding stoichiometries is discussed in terms of the mechanism involved in the formation of spectral bands. We conclude that pigment-protein interactions dominate in the determination of spectral heterogeneity with probably only minor effects on absorption associated with pigment-pigment interactions.

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.