Analysis of Some Optical Properties of a Native and Reconstituted Photosystem II Antenna Complex, CP29: Pigment Binding Sites Can Be Occupied by Chlorophyll a or Chlorophyll b and Determine Spectral Forms ² Elisabetta Giuffra, ‡,⊥ Giuseppe Zucchelli, | Dorianna Sandona `, ‡ Roberta Croce, | Daniela Cugini, ‡ Flavio M. Garlaschi, | Roberto Bassi, ‡ and Robert C. Jennings* ,| Centro CNR Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia, UniVersita ` di Milano, Via Celoria 26, 20133 Milano, Italy, and UniVersita ` di Verona, Facolta ` di Scienze MMFFNN, Biotecnologie Vegetali, Strada Le Grazie, 37134 Verona, Italy ReceiVed May 14, 1997; ReVised Manuscript ReceiVed July 28, 1997 X ABSTRACT: 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 Q y 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. Photosystems of plants are made up of a number of chlorophyll-protein complexes each of which binds many chl 1 molecules. While the outer antenna complexes of PSII and PSI bind the chemically distinct chlorophylls a and b, the core complexes contain only chl a. A detailed under- standing of energy transfer processes in the antenna and reaction centers requires knowledge of the topological organization of subunits (Bassi & Dainese, 1992; Boekema et al., 1995) and knowledge of distances between chro- mophores, mutual transition dipole orientation, and absorp- tion and fluorescence energy levels. In the case of photo- system II, progress has been made in understanding the topological organization of subunits and their spectral form composition (Hemelrijk et al., 1992; Jennings et al., 1993; Krawczyk et al., 1993; Zucchelli et al., 1994). Moreover, elucidation of the structure of the major antenna complex LHCII to a resolution of 3.7 Å (Ku ¨hlbrandt et al., 1994) has allowed identification of chlorophyll binding sites. From this structure, it is evident that many of the binding site environments are chemically distinct and that nearest neigh- bor chlorophylls are spaced by 8-15 Å. The orientation of electronic transition dipole moments of the chromophores cannot yet be obtained from the structural data, but linear dichroism analysis suggests considerable orientation hetero- geneity (Hemelrijk et al., 1992; Zucchelli et al., 1994). It has been apparent for many years that the chlorophyll proteins are spectroscopically complex. Thus, while only one, or at the most two, chemically distinct chlorophyll species are present, depending on the complex, many optical transitions (spectral forms) are commonly observed in the ² This work was supported in part by the “Piano Nazionale Biotecnologie Vegetali”, MIRAAF (Italy). * To whom correspondence should be addressed E-mail: Robert.Jennings@unimi.it. ‡ Universita ` di Verona. | Universita ` di Milano. ⊥ Present address: Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden. X Abstract published in AdVance ACS Abstracts, September 15, 1997. 1 Abbreviations: CD, circular dichroism; chl, chlorophyll; CP, chlorophyll-protein; nCP29, native protein purified from thylakoid membranes; rCP29(x), recombinant protein obtained by reconstitution in Vitro of the overxpressed apoprotein with the chromophores, with x referring to the chl a/b ratio of the reconstituted complex; DCCD, dicychlohexylcarbodiimide; FWHM, full width at half-band maximum; LHC II, light-harvesting complex of PSII; PS, photosystem. 12984 Biochemistry 1997, 36, 12984-12993 S0006-2960(97)01133-1 CCC: $14.00 © 1997 American Chemical Society