Solar energy conversion by unadapted photosystem components

We first looked at whether plant LHCIIs can pass harvested energy to purple bacterial RCs in dilute solution in the absence of complementary genetic adaptations to promote specific heterodimerisation (complexes defined as “unadapted”). On receipt of excitation energy, photochemical charge separation in the Rba. sphaeroides RC is a rapid four-step process (Fig. 1d) that produces a metastable oxidised primary electron donor (P870+) and reduced acceptor ubiquinone (Q B −); energy transfer can therefore be detected as a quenching of LHC emission accompanied by an enhancement of P870 oxidation. Although bacterial RCs and plant LHCIIs (see Methods for sources) have overlapping absorbance and emission spectra between 640 and 800 nm (Fig. 1b), no appreciable energy transfer was observed when wild-type (WT) RCs were mixed in solution with an LHCII because they have no capacity for binding to one another. The addition of purified wild-type (WT) RCs did not significantly reduce emission from LHCII (Fig. 2a) and photo-oxidative bleaching of the absorbance band of this RC’s P870 primary electron donor BChls in response to 650 nm excitation was not significantly enhanced by the addition of LHCII (Fig. 2b), which absorbs strongly at this wavelength (Fig. 1b).

Fig. 2: Energy transfer requires co-localisation of RCs and LHCs. a LHCII emission (excitation at 475 nm) and LHCI emission (excitation at 500 nm) in the absence and presence of WT RCs. The latter spectra are offset for clarity. b Data and fits for photobleaching and dark recovery of P870 absorbance for the WT RC in the absence and presence of LHCII (using variant LHCII-T). c Photobleaching and dark recovery of P870 absorbance in WT RCs in the absence and presence of LHCI (using variant Td-LHCI-Td). d Schematic of photocurrent generation on a nanostructured silver electrode; black arrows show the route of electron transfer and red arrows show energy flow. e Solution absorbance and EQE spectra for WT RCs compared with those for mixtures of WT RCs with either LHCII-T or Td-LHCI-Td. The absorbance spectra were normalised at 804 nm, while each EQE spectrum was normalised to the corresponding absorbance spectrum at the maximum of the long-wavelength P870 band. Full size image

In comparison to LHCII, the spectral overlap (J) between LHC emission and RC absorbance is ~80% larger in the case of LHCI (Fig. 1c, Supplementary Table 1) which contains a pair of “red-form” chlorophyll a that possess a charge-transfer state that mixes with the low-energy exciton state38. Although the addition of WT RCs did bring about a decrease in LHCI emission (Fig. 2a), there was no associated significant increase in RC P870 photobleaching in the presence of LHCI (Fig. 2c), leading to the conclusion that the observed emission quenching was not due to energy transfer. Protein concentrations used for the fluorescence measurements were too low (max absorbance < 0.07) for this drop in LHCI emission to be attributable to reabsorption by the added RCs, and an equivalent drop was not seen for LHCII and WT RCs at similar concentrations (Fig. 2a). As it is known that the emission quantum yield of LHCI in vitro is much more sensitive to its environment than is the case for LHCII36, the observed drop in LHCI emission on adding WT RCs is attributed to a change in its intrinsic quantum yield rather than being a signature of energy transfer.

Although no significant energy transfer was seen between these proteins in dilute solution, to establish the principle that plant LHCs can pass energy to bacterial RCs when brought sufficiently close together, mixtures of LHC and WT RC proteins were adhered to a nanostructured silver cathode and their capacity for generating photocurrents examined (see Methods). In this photoelectrochemical system (Fig. 2d) cytochrome c (cyt c) is used to “wire” charge separation in the RC to the cathode, and ubiquinone-0 (Q 0 ) shuttles electrons to the counter electrode20,39,40. Electrodes drop-cast with purified WT RCs produced a photocurrent in response to RC-specific 870 nm light and a weaker current in response to 650 nm excitation where RC absorbance is very low (Supplementary Fig. 2a). An EQE action spectrum showed good correspondence with the RC absorbance spectrum (Fig. 2e, magenta versus black), confirming that the photocurrent was attributable to light capture by the pigments of the RC. As expected, an electrode fabricated with purified LHCII failed to show any photocurrent response during 650 nm excitation of the main low-energy LHCII absorbance band (Supplementary Fig. 2a).

For electrodes fabricated from mixtures of WT RCs and LHCs, in addition to the expected RC bands the EQE spectra contained a component between 620 and 700 nm that corresponded to the low-energy absorbance band of LHCII or LHCI (Fig. 2e, green). A contribution from the high-energy Soret absorbance band of LHCII or LHCI was also observed in EQE spectra (Supplementary Figs. 3a, b and 4). This demonstrated that bacteriochlorophyll-based purple bacterial RCs can utilise chlorophyll-based plant LHCs for energy harvesting, producing charge separation and a photocurrent response, provided they are brought within Förster resonance energy transfer (FRET) distance of one another. In this case this was realised by colocalising the two proteins on the surface of a bio-photoelectrode.

Design and production of components for chimeric photosystems

In an attempt to activate chlorophyll to bacteriochlorophyll energy transfer in dilute solution, RCs and LHCs were adapted using the SpyTag/SpyCatcher protein fusion system41 as a programmable interface (see Supplementary Note 1). When mixed in solution, highly specific binding of the short SpyTag peptide to the SpyCatcher protein domain initiates autocatalysis of an isopeptide bond between the two involving aspartate and lysine residues (Supplementary Fig. 1f), producing a single, covalently locked, water-soluble protein domain41.

To adapt the RC for LHC binding an optimised version of SpyCatcher42, 106 amino acids in length (SpyCatcherΔ), was attached to the N-terminus of the RC PufL protein either directly (dubbed “RCC”) or via a four residue linker (dubbed “RC4C”) (Fig. 3a, Supplementary Table 2). Adapted RC proteins were expressed in Rba. sphaeroides (see Methods). For LHCII, Lhcb apoproteins were expressed in E. coli and mature pigment-protein monomers refolded in vitro with purified pigments43,44,45,46 (see Methods). Three LHCII proteins were designed (Fig. 3b; see Supplementary Fig. 5a for protein sequences). The first, dubbed “dLHCII”, lacked 12 dispensable N-terminal amino acids that are not resolved in available X-ray crystal structures30,31,32 and had a His-tag at its C-terminus (see Supplementary Note 1). The remaining two had either a truncated SpyTag variant (SpyTagΔ) added to the N-terminus of the truncated Lhcb1 (termed Td-dLHCII) or the full SpyTag sequence added to the C-terminus of the full Lhcb1 (termed LHCII-T) (Fig. 3b).

Fig. 3: Engineering and assembly of RC-LHC chimeras. a Construct designs for adaptation of the RC. For purification the WT RC was modified with a His-tag on PufM. b Construct designs for adaptation of LHCII. The control LHCII was truncated at its N-terminus (dLHCII—see text) and was His-tagged at its C-terminus. c Construct designs for adaptation of LHCI which is an Lhca1/Lhca4 heterodimer. For b and c protein sequences are given in Supplementary Fig. 5a. d Sucrose density gradient fractionation of RCs (red bands) and LHCIIs (green bands). RC-LHCII chimeras migrate to a lower position in gradients than either RC or LHCII monomers, with no dissociation into components. e Blue NativePAGE showing the formation of high molecular weight products by mixing LHCI-Td or Td-LHCI-Td with RCC (see Supplementary Fig. 7a for the full gel with more combinations). The multiple bands seen for the high molecular weight products are likely to be due to conformational heterogeneity. f Sucrose density gradient fractionation of RCs (red bands) and LHCIs (green bands). LHCI#RC chimeras and larger RC#LHCI#RC chimeras migrate to lower positions than either RCs or LHCI. g TEM images of an equimolar mixture of the WT RC and dLHCII (top/left), the LHCII#RC chimera (top/right), the LHCI#RC chimera (bottom/left) and the RC#LHCI#RC chimera (bottom/right). Additional images shown in Supplementary Fig. 17. Scale bar represents 50 nm. h Molecular model of the LHCII#RC chimera. The RC (maroon) N-terminally adapted with SpyCatcherΔ (blue) is covalently linked to LHCII (green) C-terminally adapted with SpyTag (yellow). Cofactor colours are as described in Supplementary Fig. 1. i Molecular models of the LHCI#RC and RC#LHCI#RC chimeras. Colours as for panel h and Supplementary Fig. 1. Full size image

Adapted heterodimeric LHCI proteins (Fig. 3c; see Supplementary Fig. 5a for protein sequences) were also refolded from apoproteins expressed in E. coli34,38,47,48. This involved mixing SpyTagΔ-adapted Lhca4 protein (Td-L4) with either unadapted Lhca1 protein (L1) or SpyTagΔ-adapted Lhca1 protein (Td-L1) to produce LHCI either singly or doubly modified with SpyTagΔ (termed LHCI-Td and Td-LHCI-Td, respectively). This enabled the creation of chimeras between LHCI and either one or two RCs (see further details in Supplementary Note 1).

Self-assembly of RC-LHC chimeras

Following ultracentrifugation, purified RCs and LHCIIs could be visualised on sucrose density gradients as either a red or green band, respectively (Fig. 3d, gradients 1 and 2), and these two proteins also migrated separately in gradients loaded with a mixture with only either the SpyTag or SpyCatcher adaptations (Fig. 3d, gradients 3 and 4). In contrast, mixing any SpyCatcherΔ-adapted RC with any SpyTag(Δ)-adapted LHCII produced a product, dubbed a “chimera”, that migrated further than either monomeric protein. The two examples shown in Fig. 3d (gradients 5 and 6) are chimeras from a RC4C/Td-dLHCII mix (dubbed “RC#LHCII”) and from a RCC/LHCII-T mix (dubbed “LHCII#RC”). The symbol “#” denotes the spontaneously formed SpyCatcher/SpyTag interface domain. Chimera formation could also be detected on a native blue gel (Supplementary Fig. 6a). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) combined with western blotting using anti-His antibodies confirmed that chimera self-assembly was due to the formation of a covalent bond between the SpyTag(Δ)-adapted Lhcb1 polypeptide of LHCII and the SpyCatcherΔ-adapted PufL polypeptide of the RC (Supplementary Fig. 6b, Supplementary Note 2). The reaction half-time for chimera formation varied between 10 and 90 min depending on the particular combination of adapted RC and LHCII (detailed in Supplementary Note 3).

LHCI-RC chimeras could also be assembled by incubation of LHCI-Td or Td-LHCI-Td with a threefold excess of RCC. This again produced higher molecular weight products that could be separated from unreacted RCs on blue native gels (Fig. 3e). As designed, assembly of RCC with doubly adapted Td-LHCI-Td complexes produced higher molecular weight products than with singly adapted LHCI-Td complexes (Fig. 3e, right). Equivalent results were obtained with LHCI adapted with the full SpyTag and also with RC4C (Supplementary Fig. 7a). Analysis by SDS-PAGE and western blotting showed that chimera self-assembly was due to formation of a covalent bond between the SpyCatcherΔ-adapted PufL of the RC and Lhca4 of a singly SpyTagΔ-adapted LHCI (to form chimera LHCI#RC) or Lhca4 and Lhca1 of a doubly SpyTagΔ-adapted LHCI (to form chimera RC#LHCI#RC) (Supplementary Fig. 7b). Sucrose density gradient ultracentrifugation (Fig. 3f) showed that LHCI#RC chimeras (gradient 5) were clearly larger than LHCI alone (gradients 2–4) or unadapted RCs (gradients 1, 3 and 4), and RC#LHCI#RC chimeras (gradient 6) were larger again.

Covalent locking of the structure enabled purification of all LHCI-RC and LHCII-RC chimeras by a combination of nickel affinity and size-exclusion chromatography, absorbance spectroscopy being used to identify fractions containing protein oligomers with the designed molar ratio (Supplementary Fig. 8).

A change in protein morphology on chimera formation could be observed by transmission electron microscopy (TEM). Images of a mix of unadapted WT RCs and dLHCII showed a large number of monodispersed, regularly sized objects of <10 nm diameter (Fig. 3g, top/left), whereas images of the purified LHCII#RC chimera revealed two-domain objects (Fig. 3g, top/right). The purified LHCI#RC and RC#LHCI#RC chimeras presented as objects with a more elongated morphology owing to the presence of one or two RCs and the heterodimeric LHCI (Fig. 3g, bottom). Molecular models of these chimeras, based on available X-ray crystal structures for the RC, LHCII, LHCI and SpyCatcher/Tag, are shown in Fig. 3h, i.

Chlorophyll to bacteriochlorophyll energy transfer

In solution, LHCII emission was quenched within each chimera in comparison to a control sample formed from an equivalent mix of the SpyTag-adapted LHCII and WT RCs (Fig. 4a; see spectra and other combinations in Supplementary Fig. 9). This was indicative of energy transfer, likely through a FRET mechanism at the distances implied by the chimera models (Fig. 3h, i), that was activated in these proteins in dilute solution by physically linking the RC to the LHCII. These trends, observed with 650 nm excitation, were also seen in data on the same complexes obtained with other three excitation wavelengths, with no variation in emission spectrum line shape (Supplementary Fig. 9). As well as being diagnostic of correctly refolded LHCII proteins, this lack of dependence of emission spectrum on excitation wavelength showed that the reduction in LHCII emission on chimera formation was not due to parasitic RC absorbance, which would be expected to be wavelength dependent (and also seen when WT RCs were mixed with each LHCII).

Fig. 4: Energy transfer in chimeras in solution and on surfaces. a Emission at 682 nm from (left) the Td-dLHCII protein alone (grey), after addition of a twofold excess of WT RCs (green) and in a LHCII#RC chimera (cyan) formed on mixing with a twofold excess of RCC, and (right) equivalent data for LHCII-T. b Data and fits for photobleaching and dark recovery of P870 absorbance in RCC, a 1:1 RCC plus dLHCII mixture and the LHCII#RC chimera. c Emission from (left) LHCI-Td alone (grey), after addition of a threefold excess of WT RCs (green) and in a LHCI#RC chimera (navy) formed on mixing with a threefold excess of RCC, and (right) equivalent data for Td-LHCI-Td. Error bars in a and c represent standard deviations from six repeats (three technical repeats of two biological repeats) and data points are shown as overlaid circles. d Data and fits for photobleaching and dark recovery of P870 absorbance in RCC and the two RC-LHCI chimeras. e Solution absorbance and EQE spectra for WT RCs compared with those for the two LHCII-RC chimeras. f Solution absorbance and EQE spectra for WT RCs compared with those for the two RC-LHCI chimeras. g Schematic of adsorption of independent RC (red) and LHCII (green) complexes on an electrode. Yellow arrows indicate possible energy transfer connections. h Equivalent schematic of adsorption of RC#LHCII chimeras. i Simulated apparent ET efficiencies as a function of packing density for independent RC and LHCII proteins or the LHCII#RC chimera. Error bars represent standard deviations from ten simulation repeats. Individual data points are represented by circles or squares. Full size image

To determine the fate of transferred energy, measurements of RC P870 photooxidation in response to 650 nm excitation were carried out on the LHCII#RC and RC#LHCII chimeras and fitted to a simple interconversion reaction (Eq. (1); all parameters are summarised in Supplementary Table 3). Bleaching of 870 nm absorbance was stronger in LHCII#RC chimeras than in controls comprising the RCC protein alone or a mixture of RCC with unadapted dLHCII complexes (Fig. 4b). The same was found for the RC#LHCII chimera (Supplementary Fig. 10a). Hence, decreased emission by the LHCII energy donor was accompanied by enhanced photooxidation of the RC energy acceptor, confirming energy transfer between the two proteins in solution that was switched on only after linking them by the SpyCatcher/Tag domain.

Turning to LHCI, a greater reduction of LHCI emission was seen on forming either LHCI#RC or RC#LHCI#RC chimeras than after mixing the same adapted LHCI proteins with WT RCs (Fig. 4c; and other combinations are shown in Supplementary Fig. 11). This effect was again seen to be independent of excitation wavelength (Supplementary Fig. 11a) showing it was not due to the absorbance of excitation light by the tethered RC(s). This emission quenching was accompanied by significant enhancement of P870 photooxidation in LHCI chimeras with one or two RCC, compared to that seen with RCC alone (Fig. 4d), confirming energy transfer. Doubly modified RC#LHCI#RC complexes showed less P870 bleaching than LHCI#RC complexes due to two tethered RCs competing for the exciton reservoir rather than one (see below).

Purified chimeras were also adhered to nanostructured silver electrodes to test their functionality. All were able to generate photocurrents, showing that dynamic interactions between the RC, cyt c and ubiquinone at the electrode surface, required for the generation of a photocurrent, were not obstructed by attaching the RC to LHCII or LHCI. All EQE action spectra recorded for chimeras exhibited low-energy (Fig. 4e, f) and high-energy chlorophyll bands (Supplementary Figs. 3c, d and 4, green shading) indicating photocurrent generation powered by LHC absorbance.

Energy transfer efficiency in chimeras

Apparent efficiencies of energy transfer from LHCII or LHCI to the RC in solution were estimated either from data on emission of the LHC energy donor (E FL ) or from data on photobleaching of the RC energy acceptor (E P870 ) (see Methods, Eqs. (2)–(4)). Efficiency E FL was based on the additional quenching of LHC emission in a chimera relative to that in a compositionally matched mixture of the relevant LHC variant and WT RCs (Eqs. (2) and (3)) or additional quenching in an LHC/WT RC mixture relative to that in a concentration-matched LHC-only sample. Efficiency E P870 was based on the enhanced rate of RC P870 photobleaching in a chimera relative to a matched RC-only control (Eq. 4).

Values of E P870 calculated from experimental data are shown in Table 1. The efficiency of energy transfer was low in mixtures of WT RCs with SpyTag-adapted LHCIIs or LHCIs, consistent with expectations for a dilute (500 nM) solution of two proteins with no propensity to associate (see Supplementary Fig. 12 and Supplementary Table 4 for other control combinations). In marked contrast, E P870 was over 20% in the corresponding RC-LHCII or RC-LHCI chimera (Table 1). For all chimeras the value of E FL derived from LHC emission data was in excellent agreement with the values of E P870 derived from RC absorbance data (Table 1). This correspondence between independently determined efficiencies from separate data sets reinforced the conclusion that energy transfer was taking place from the plant LHCs to the bacterial RCs within the chimera.

Table 1 Apparent energy transfer efficiencies and associated parameters. Full size table

“On electrode” apparent energy transfer efficiencies (E electrode ) were also determined from the EQE action spectra, as described in Methods. In general, values of E electrode were higher than either estimate of energy transfer efficiency in solution (Table 1). This was particularly striking for mixtures of WT RCs and SpyTag(Δ)-adapted LHCII or LHCI (shown schematically in Fig. 4g) where energy transfer in solution had a very low apparent efficiency. However, for the RC/LHCII chimeras in particular the value of E electrode was also substantially higher than E P870 or E FL (Table 1, Fig. 4h), suggesting that adhering the chimeras to a surface turned on inter-chimera ET that supplemented the intra-chimera ET observed in solution. This effect was less pronounced for the RC/LHCI chimeras, particularly for complex RC#LHCI#RC where there were already two RCs per LHCI antenna (Table 1).

To examine whether the benefits of pre-linking RCs and LHCs in a chimera would be seen across a range of surface packing densities, a 2D Monte Carlo simulation was carried out as detailed in Supplementary Notes 4 and 5 (and summarised in Supplementary Fig. 13). In this either LHCII#RC chimeras or a mixture of LHCII-T and WT RC proteins were represented as hard-discs on a 2D surface and centre-to-centre distances calculated as a function of packing density. The outcome of this simulation was an apparent energy transfer efficiency (E sim ) based on how protein packing densities affected overall inter-protein distances. In the high packing regime, E sim was in good agreement with the slightly higher E electrode determined for chimeras than for a mixture of unadapted proteins (Fig. 4i, right). As the packing density dropped to a low value (right to left in Fig. 4i), E sim for the chimeras gradually declined to around the values for E P870 and E FL estimated for the LHCII#RC chimera in solution (22.8%/19.6%). In contrast, E sim for the protein mixture declined steeply to less than 2% at the lowest packing density, again in agreement with estimates of E P870 and E FL for the protein mixture in solution (1.2%/0.8%). This reinforced the conclusion that pre-tethering of the RC and LHCII protein into a chimera brought an added benefit even under conditions where co-localisation of the proteins on a surface switched on energy transfer between the two irrespective of tethering. Presumably pre-tethering can mitigate against situations where, for example, formation of RC-rich or LHCII-rich sub-domains and sub-optimal mixing can lead to some proteins being outside the FRET distance (Supplementary Fig. 14, marked with blue triangles).