Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna |
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Authors: | David J.K. Swainsbury Kaitlyn M. Faries Dariusz M. Niedzwiedzki Elizabeth C. Martin Adam J. Flinders Daniel P. Canniffe Gaozhong Shen Donald A. Bryant Christine Kirmaier Dewey Holten C. Neil Hunter |
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Affiliation: | 1. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK;2. Department of Chemistry, Washington University, St. Louis, MO 63130-4889, USA;3. Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA |
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Abstract: | The light-harvesting 2 complex (LH2) of the purple phototrophic bacterium Rhodobacter sphaeroides is a highly efficient, light-harvesting antenna that allows growth under a wide-range of light intensities. In order to expand the spectral range of this antenna complex, we first used a series of competition assays to measure the capacity of the non-native pigments 3-acetyl chlorophyll (Chl) a, Chl?d, Chl?f or bacteriochlorophyll (BChl) b to replace native BChl?a in the B800 binding site of LH2. We then adjusted the B800 site and systematically assessed the binding of non-native pigments. We find that Arg?10 of the LH2 β polypeptide plays a crucial role in binding specificity, by providing a hydrogen-bond to the 3-acetyl group of native and non-native pigments. Reconstituted LH2 complexes harbouring the series of (B)Chls were examined by transient absorption and steady-state fluorescence spectroscopies. Although slowed 10-fold to ~6?ps, energy transfer from Chl?a to B850 BChl?a remained highly efficient. We measured faster energy-transfer time constants for Chl?d (3.5?ps) and Chl?f (2.7?ps), which have red-shifted absorption maxima compared to Chl?a. BChl?b, red-shifted from the native BChl?a, gave extremely rapid (≤0.1?ps) transfer. These results show that modified LH2 complexes, combined with engineered (B)Chl biosynthesis pathways in vivo, have potential for retaining high efficiency whilst acquiring increased spectral range. |
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Keywords: | LH2 Bacteriochlorophyll Chlorophyll Light harvesting Ligand binding Protein engineering |
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