title: 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.
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 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.
authors.affiliation: Centro CNR Biologia Cellulare e Molecolare delle Piante, Dipartimento di Biologia, Università di Milano, Via Celoria 26, 20133 Milano, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Giuffra
authors.forename: G
authors.initials: G
authors.lastname: Zucchelli
authors.forename: D
authors.initials: D
authors.lastname: Sandonà
authors.forename: R
authors.initials: R
authors.lastname: Croce
authors.forename: D
authors.initials: D
authors.lastname: Cugini
authors.forename: F M
authors.initials: FM
authors.lastname: Garlaschi
authors.forename: R
authors.initials: R
authors.lastname: Bassi
authors.forename: R C
authors.initials: RC
authors.lastname: Jennings
mesh_terms: D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002734:Chlorophyll / Q000378:metabolism*; D000077194:Chlorophyll A; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008027:Light; D045342:Light-Harvesting Protein Complexes; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045332:Photosystem II Protein Complex; D010860:Pigments, Biological / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
chemical_list: D001059:Apoproteins; C069660:CP29 light harvesting complex; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010860:Pigments, Biological; D011994:Recombinant Proteins; D002734:Chlorophyll; C037184:chlorophyll b; D000077194:Chlorophyll A