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Volume 270, Number 11, Issue of March 17, 1995 pp. 5793-5804
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Reconstitution of the Bacterial Core Light-harvesting Complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with Isolated - and -Polypeptides, Bacteriochlorophyll a, and Carotenoid

(Received for publication, October 12, 1994; and in revised form, January 4, 1995)

Christine M. Davis,  Peggy L. Bustamante,  Paul A. Loach

Methodology has been developed to reconstitute carotenoids and bacteriochlorophyll a with isolated lightharvesting 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 a 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 a 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.



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