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.
Highlights
The phototrophic purple bacterium Rhodobacter sphaeroides (Rba. sphaeroides) utilises light to generate ATP within specialised membrane compartments [1,2] that contain four major complexes, light-harvesting complex 2 (LH2), reaction-centre light-harvesting 1-PufX complex (RC-LH1PufX), cytochrome bc1 and ATP synthase [3,4]
To gain further insight into (B)Chl binding to the B800 site, we examined the crystal structure of Rbs. acidophilus LH2 [14]
This structure shares 45% and 66% sequence identity with the α and β polypeptides of Rba. sphaeroides LH2, respectively and key conserved residues are essential for correct binding of bacteriochlorophyll a (BChl a) in the Rba. sphaeroides complex [17,20,73]
Summary
The phototrophic purple bacterium Rhodobacter sphaeroides (Rba. sphaeroides) utilises light to generate ATP within specialised membrane compartments [1,2] that contain four major complexes, light-harvesting complex 2 (LH2), reaction-centre light-harvesting 1-PufX complex (RC-LH1PufX), cytochrome bc and ATP synthase [3,4]. Light is absorbed by either the LH2 or LH1 antenna, arranged in protein-rich arrays, and excitation energy is transferred within picoseconds to the RC, where charge separation drives the reduction of quinone to quinol and the oxidation of cytochrome c2 [5,6,7,8,9,10]. These products are utilised by the cytochrome bc complex, which resides in a locally lipid-rich region, to re-reduce oxidised cytochrome c2 and oxidise quinol to quinone [3,4,11,12]. Another interaction of note is a BBA-Bioenergetics1860(2019)209223
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