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 pigmentbinding 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.In higher-plant chloroplasts, many pigment-binding proteins are inserted into the thylakoid membrane and organized into multisubunit complexes called photosystems. Photosystems (PS) 1 catalyze light absorption, the use of the excitation energy in transmembrane electron transport leading to the synthesis of ATP and NADPH. PS I and II are both composed of a chlorophyll a binding core complex and a chlorophyll a and b binding antenna. A detailed understanding of energy transfer processes in antenna and reaction centers requires knowledge of the distances between chromophores, of the mutual orientation between dipole transitions, and of the absorption and fluorescence energy levels and their distribution within the photosystems. In the case of PSII, considerable insight has been obtained from the topological organization of subunits (1-3) and from their spectral form decomposition (4, 5). Elucidation of the major antenna complex LHCII structure to 3.4 Å resolution (6) showed that many of the binding site environments are chemically distinct and that nearest-neighbor chlorophylls (Chl) are spaced 9-13 Å apart (center-center distance). The orientation of electronic transition dipole moments of the chromophores cannot yet be obtained from structural data, although linear dichrois...

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