Isolation and Characterization of the B798 Light-Harvesting Baseplate from the Chlorosomes of Chloroflexus aurantiacus ² Gabriel A. Montan ˜o, § Hsing-Mei Wu, ‡ Su Lin, ‡ Daniel C. Brune, ‡ and Robert E. Blankenship* ,‡ Department of Chemistry & Biochemistry, Center for the Study of Early EVents in Photosynthesis, and Graduate Program in Molecular and Cellular Biology, Arizona State UniVersity, Tempe, Arizona 85287-1604 ReceiVed March 4, 2003; ReVised Manuscript ReceiVed July 5, 2003 ABSTRACT: The B798 light-harvesting baseplate of the chlorosome antenna complex of the thermophilic, filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus has been isolated and character- ized. Isolation was performed by using a hexanol-detergent treatment of freeze-thawed chlorosomes. The isolated baseplate consists of Bchl a, -carotene, and the 5.7 kDa CsmA protein with a ratio of 1.0 CsmA protein/1.6 Bchl a/4.2 -carotenes. The baseplate has characteristic absorbance at 798 nm as well as carotenoid absorbance maxima at 519, 489, and 462 nm. The energy transfer efficiency from the carotenoids to the Bchl a is 30% as measured by steady-state and ultrafast time-resolved fluorescence and absorption spectroscopies. Energy equilibration within the Bchl a absorbing regions exhibits ultrafast kinetics. Circular dichroism spectroscopy shows no evidence for excitonically coupled Bchl a pools within the 798 nm region. The green photosynthetic bacteria are anoxygenic pho- totrophs that contain an antenna structure known as the chlorosome (1, 2). Chlorosomes transfer energy to the photosynthetic reaction centers. Both the green sulfur and filamentous anoxygenic phototrophic (FAP) 1 bacteria (3) contain chlorosomes with similar architecture; however, the pigment content and overall makeup of the chlorosome are dependent on the species (1). Green sulfur bacterial chlo- rosomes are attached to Fenna-Matthews-Olson (FMO) antenna proteins that serve as an intermediary in energy transfer to the photosynthetic reaction centers (1, 2). FAP bacteria lack the FMO complex, and the chlorosomes instead are attached directly to the integral membrane photosynthetic complexes (1). The chlorosomes of the FAP bacterium Chloroflexus aurantiacus (150 × 50 × 10 nm) sit on the cytoplasmic side of the membrane, efficiently absorb light energy, and funnel it to the reaction center via the B808- 866 integral membrane antenna complex (1, 4, 5). The chlorosome is an unusual antenna complex in that it contains a very high pigment-to-protein ratio and is believed to rely primarily on pigment-pigment interactions instead of pigment-protein interactions (1). Chlorosomes contain a large amount of bacteriochlorophyll c, d, or e, carotenoids, and a small amount of Bchl a along with proteins. The complex is surrounded by a monolayer membrane composed primarily of monogalactosyl diglyceride (MGDG) (1, 6, 7). Pigment-pigment aggregation in the chlorosome of C. aurantiacus causes a spectral shift from 670 nm (monomeric Bchl c) to 742 nm (aggregated Bchl c)(1). The aggregates organize themselves into rod-shaped structures that fill the interior of the chlorosome (4, 8) and pass energy to the B798 nm baseplate (6, 7) that serves as an intermediary in energy transfer to the membrane antenna and reaction center. The baseplate is believed to be a pigment-protein complex that is situated at the base of the chlorosome and connects it to the membrane-associated pigment-protein complexes (1, 7). Electron microscopy has shown a crystalline structure on the order of 6 nm periodicity that creates the attachment site for the chlorosome to the membrane (4). This may be due to the so-called B798 nm baseplate complex, although this has not been demonstrated. The location and function of the chlorosomal proteins is still under debate. Purified C. aurantiacus chlorosomes contain three major protein componentssCsmA, CsmM and CsmNsof molecular masses 5.7, 11, and 18 kDa, respec- tively, and a minor component of mass 5.8 kDa (1, 7). The first three proteins have been localized to the lipid envelope by gold labeling electron microscopy (9); however, their specific functional roles have not been determined. None of the proteins were found to be specifically localized to the cytoplasmic membrane side of the chlorosome where the baseplate is presumably located (9) using antibody methods, although fractions of the CsmM and CsmN peptides were observed in this region. Proteolytic digestion experiments and CD spectroscopy have suggested the involvement of proteins in determining the overall chlorosome structure, specifically implicating the 5.7 kDa CsmA protein in the organization of the Bchl c aggregates (10-13). However, the interpretation that the 5.7 ² Supported by U.S. Department of Energy Grant DE-FG03- 01ER15214; Student Financial support for Gabriel A. Montan ˜ o through NSF Research Training Grant for Optical Biomolecular Devices #9602258. * Corresponding author: Telephone (480) 965-1439; fax (480) 965- 2747; e-mail- blankenship@asu.edu. ‡ Department of Chemistry & Biochemistry. § Graduate Program in Molecular and Cellular Biology. 1 Abbreviations: Bchl, bacteriochlorophyll; MGDG, monogalactosyl diglyceride; SPC, single photon counting; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; FAP, filamentous anoxy- genic phototroph. 10246 Biochemistry 2003, 42, 10246-10251 10.1021/bi034350k CCC: $25.00 © 2003 American Chemical Society Published on Web 08/07/2003