RECONSTITUTION OF THE BACTERIAL CORE LIGHT-HARVESTING COMPLEXES OF RHODOBACTER-SPHAEROIDES AND RHODOSPIRILLUM-RUBRUM WITH ISOLATED ALPHA-POLYPEPTIDES AND BETA-POLYPEPTIDES, BACTERIOCHLOROPHYLL-A, AND CAROTENOID

Methodology has been developed to reconstitute carotenoids and bacteri ochlorophyll a with isolated light-harvesting complex I (LHI) polypept ides of both Rhodobacter sphaeroides and Rhodospirillum rubrum, Recons titution techniques first developed in this laboratory using the LHI p olypeptides of R. rubrum, R. sphaeroides, and Rhodobacter capsulatus r eproduced bacteriochlorophyll a spectral properties characteristic of LHI complexes lacking carotenoids. In this study, carotenoids are supp lied either as organic-solvent extracts of chromatophores or as thin-l ayer chromatography or high performance liquid chromatography-purified species. The resulting LHI complexes exhibit carotenoid and bacterioc hlorophyll a spectral properties characteristic of native LHI complexe s of carotenoid-containing bacteria. Absorption and circular dichroism spectra support the attainment of a native-like carotenoid environmen t in the reconstituted LHI complexes. For both R. sphaeroides- and R. rubrum-reconstituted systems, fluorescence excitation spectra reveal a ppropriate carotenoid to bacteriochlorophyll a energy-transfer efficie ncies based on comparisons with the in vivo systems. In the case of R, rubrum reconstitutions, carotenoids afford protection from photodynam ic degradation. Thus, carotenoids reconstituted into LHI exhibit spect ral and functional characteristics associated with native pigments. He terologous reconstitutions demonstrate the applicability of the develo ped assay in dissecting the molecular environment of carotenoids in Li ght-harvesting complexes.