| Light-harvesting complexes (LHCs) are nuclear-encoded integral proteins in thylakoid membranes that collect and regulate delivery of exciton energy to photosynthetic reaction centers. Pigments, chlorophylls (Chl) a and b along with active xanthophylls, are required co-factors in assembly and function of LHCs. To investigate the in vivo site and mechanism of assembly of LHCs, the model organism, Chlamydomonas reinhardtii, was used for this study. Chl a and Chl b differ only in oxidation of the 7-methyl group of Chl a to a formyl group. Chlorophyll(ide) a oxygenase, the enzyme responsible for the oxidation, is membrane-associated and located on the inner envelope and thylakoid membranes, as shown by western blotting and immunoelectron microscopy. Conversion of the methyl group may be initiated by a radical, located on a conserved tyrosine, as shown by electron paramagnetic resonance. The ability of Chl b to form stronger coordination bonds with amino acid sidechains of the apoproteins of LHCs shifts equilibrium positions of complex formation toward assembly of LHCs in chloroplasts. In C. reinhardtii, Chl b is required for the import and retention of LHCs by the chloroplast. If Chl b is not available during import, assembly does not proceed and LHC apoproteins are shunted to cytoplasmic vacuoles. Molecular modeling of a highly conserved amino acid region within the apoprotein of LHCs, Glu-X-X-His/Asn-X-Arg, suggested that this motif may bind two molecules of Chl. The prediction was confirmed experimentally, which suggests that this motif may bind Chl during the import of LHCs. To indicate the importance of the sequence, it was designated a "retention motif." During early development of the thylakoid system in chloroplasts, the site of assembly of LHCs is on a low-density membrane (inner envelope). These LHCs are functionally coupled to reaction centers within seconds after assembly. |
