Abstract
Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll alpha with isolated light-harvesting complex I (LHI) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum. Reconstitution techniques first developed in this laboratory using the LHI polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll alpha spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supplied either as organic-solvent extracts of chromatophores or as thin-layer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacteriochlorophyll a spectral properties characteristic of native LHI complexes of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environment in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal appropriate carotenoid to bacteriochlorophyll alpha energy-transfer efficiencies based on comparisons with the in vivo systems. In the case of R. rubrum reconstitutions, carotenoids afford protection from photodynamic degradation. Thus, carotenoids reconstituted into LHI exhibit spectral and functional characteristics associated with native pigments. Heterologous reconstitutions demonstrate the applicability of the developed assay in dissecting the molecular environment of carotenoids in light-harvesting complexes.
Highlights
Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll a with isolated lightharvesting complex I (LID) polypeptides of both Rhodobacter sphaeroides and Rhodospirillum rubrum
Reconstitution techniques first developed in this laboratory using the LID polypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus reproduced bacteriochlorophyll a spectral properties characteristic of LID complexes lacking carotenoids
These results suggested that the LHI reconstitution techniques first developed using the IX- and l3-polypeptides of R. rubrum (Parkes-Leach et al, 1988) and subsequently using LHI polypeptides of R. sphaeroides and Rhodobacter capeulatus (Loach et al, 1994) could be extended to include carotenoids
Summary
Vol 270, No 11, Issue of March 17, pp. 5793-5804, 1995 Printed in U.S.A. Reconstitution of the Bacterial Core Light-harvesting Complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with Isolated a- and 13-Polypeptides, Bacteriochlorophyll a, and Carotenoid*. Specificity of carotenoid binding has been studied by incorporation of native or nonnative carotenoids into reaction centers (Boucher et al, 1977; Agalidis et al, 1980; Chadwick and Frank, 1986) or into LH complexes of carotenoidless strains (Davidson and Cogdell, 1981; Noguchi et al, 1990) In such reconstitution experiments, isolated complexes or lyophilized chromatophores were first incubated with carotenoid introduced in organic solvent. During development of the methodology for preparing LHI structural subunits from R. rubrum and Rhodopseudomonas oiridis, the addition of carotenoid to carotenoid-depleted systems resulted in partial restoration of wild-type spectra (Miller et al, 1987; Parkes-Loach et al, 1994). We report here the successful reconstitution of the IX- and l3-polypeptides of both R. sphaeroides or R. rubrum with BChlj! and the other native pigment of LHI complexes, the carotenoid
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