Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

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.

Carotenoid-to-(bacterio)chlorophyll energy transfer in LH2 antenna complexes from Rba. sphaeroides reconstituted with non-native (bacterio)chlorophylls

“…It is noticeable that k ET800 for Sphe S 1 → BChl b Q y is an apparent outlier from the overall trend. Our past studies demonstrated that this pigment correctly binds within the B800 site (Swainsbury et al 2019) so this result is rather surprising. However, in Fig.…”

“…sphaeroides cells lacking the CrtA gene [reported previously in (Chi et al 2014)] were grown under 30 μmol photons s −1 m −2 illumination from OSRAM classic 116 W halogen bulbs in M22+ medium (Hunter and Turner 1988) supplemented with 0.1% w/v casamino acids. B800-B850 LH2 was purified by ion-exchange chromatography followed by size-exclusion as previously described (Swainsbury et al 2019). To generate a set of LH2 complexes with alternative (B)Chls bound to the B800 site, purified LH2 was exchanged into pH 5.0 buffer containing lithium dodecyl sulfate (LDS).…”

“…Subsequently, B850 LH2 was incubated in excess of alternative (B)Chls (Chl a, Chl d, acChl a and BChl b) that readily bind to the vacant B800 site yielding complexes with new spectral features. For a detailed protocol see (Swainsbury et al 2019).…”

“…2. Energy transfer between non-native (B)Chls and B850 BChl a was extensively elaborated in past study (Swainsbury et al 2019) and will not be discussed here. The main focus of this investigation is to determine how the overall Sphe-to-B850 BChl a energy transfer is affected by incorporating alternative (B) Chls into the B800 site.…”

“…LH2 complexes from Rba. sphaeroides and Rhodoblastus acidophilus with alternative B800 pigments have previously been used to study energy transfer between the B800 and B850 (B)Chls and B850 LH2 complexes have previously been used to elucidate the role of B800 BChl a in carotenoid → B850 energy transfer (Bandilla et al 1998;Herek et al 2000;Linnanto and Korppi-Tommola 2002;Macpherson et al 2001;Swainsbury et al 2019). The primary focus of those studies was to elaborate excitation energy transfer between those (non-)native (B)Chls and B850 BChl a and to test the viability of in vivo pigment replacement to expand spectral coverage via protein engineering.…”