Mimicking Photosystem I with a Transmembrane Light Harvester and Energy Transfer‐Induced Photoreduction in Phospholipid Bilayers

Abstract Photosystem I (PS I) is a transmembrane protein that assembles perpendicular to the membrane, and performs light harvesting, energy transfer, and electron transfer to a final, water‐soluble electron acceptor. We present here a supramolecular model of it formed by a bicationic oligofluorene 12+ bound to the bisanionic photoredox catalyst eosin Y (EY2−) in phospholipid bilayers. According to confocal microscopy, molecular modeling, and time dependent density functional theory calculations, 12+ prefers to align perpendicularly to the lipid bilayer. In presence of EY2−, a strong complex is formed (Ka=2.1±0.1×106  m −1), which upon excitation of 12+ leads to efficient energy transfer to EY2−. Follow‐up electron transfer from the excited state of EY2− to the water‐soluble electron donor EDTA was shown via UV–Vis absorption spectroscopy. Overall, controlled self‐assembly and photochemistry within the membrane provides an unprecedented yet simple synthetic functional mimic of PS I.


Introduction
In nature, photosynthetic organisms absorb sunlight to convert it into high-energyc hemicals used as bioenergy carriers. In order to do so, they arrange severalp rotein super complexes with preciselyo riented chromophores in phospholipidm embranes. [1][2][3] One example is photosystemI(PS I) whichi ss urrounded by multiple units of the protein light harvesting complexes I( LHC I) to harvest sunlight in the UV and visible range of the solar spectrum to funnelt he photon energy to the reaction centeri np hotosystem I( PS I). [1] Light energy transfer within the membrane is enabled by orientation control of numerous light harvesting chromophores within the membrane and with respectt ot he energy accepting reactionc enter. [1] The reaction center itself is ar ed light-absorbing chlorophyll dimer which triggersm ultistepe lectron transfer reactions in the phospholipidm embrane to af inal electron acceptor. [1][2][3][4][5] Synthetic self-assembliesa re aimed at mimicking functions of cells and photosynthesis. [6][7][8] In particular, phospholipidm embranesa nd vesicles (e.g. liposomes) can serve as as caffold for mimicking cellular compartmentalization, [9][10][11] light harvesting, [12] membrane interactions, [13,14] transmembrane electron transfer, [10,[15][16][17][18][19] and co-assemblyo fp hotosensitizers with electron relays and catalysts. [20][21][22][23] In very rare cases the assembly of chromophores at phospholipidm embranes enabled for light-induced energy and electron transfer. [24] Self-assembled transmembrane molecular wires were able to achieve electron transfer across artificial and naturalp hospholipid membranes, thoughi nt he absence of light. [25][26][27][28] Liposomes doped with transmembrane electron transferringc hromophores coupled to proton and ion transfer lead to pH andc oncentrationg radients across membranes. [26][27][28] One common design principle for membrane-spanning molecules it that they shall comprise both ac entral hydrophobic and one or two terminalh ydrophilic groups. With two end-groups, the distance between these hydrophilic groups should matcht he thickness of the lipid bilayer,a sd istance mismatch tends to lower membrane stability. [29][30][31][32][33] In this study,w ec onstructed an artificial, biomimetic analogue of photosystem Ib ased on ar igid, oligofluorene chromophore that precisely self-assembles perpendicularly to phospholipid bilayers. We chose here ar igid, symmetrical oligofluorene core composed of eight conjugated aromatic rings, directly connected to two terminal, hydrophilic trimethylammonium anchoring groups. The designed oligo-fluorene 1 2 + + is depicted in Scheme 1. The ammonium groups are separated by ad istance of 3.5 nm, which fits best with typical thicknesses of phospholipid bilayers (vide infra). [33] Upon light absorption, this oligofluorene funnelst he photon energy into an energy acceptorf inally capable of transferring electrons at the watermembrane interface.

Results and Discussion
The synthesis of 1(PF 6 ) 2 was performed in four steps described in the Supporting Information. Am olecular dynamics model of 1 2 + + in ap hospholipid bilayer (Figure 1a)c onfirmedt hat the 3.5 nm distance between the ammonium groups fits ideally with the 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine( DPPC) membrane thickness of 3.1-3.4 and 3.4-3.7 nm, respectively. [34,35] In organic solvent, 1(PF 6 ) 2 absorbs at 358 nm in methanol, and itsh ydrophobic core molecule 2 (Scheme 2) absorbs at slightly higher energy in chloroform (349 nm, see Ta ble 1). In spite of their similar emission maxima(% 400 nm) and stokes shifts (48 vs. 44 nm, respectively), the molar absorption coeffi-cient (e)o f1 2 + + in methanolw as found significantly higher than that of 2 in chloroform (16 10 4 m À1 cm À1 vs. 6.8 10 4 m À1 cm À1 )s uggesting different types of excited states. Upon incorporation into liposomes neither 1 2 + + nor 2 experienced significant spectroscopicc hanges comparedt oo rganic Scheme1.Light absorption by 1 2 + + is followed by energy transfer to eosin Y (EY 2À ,inred) andsubsequent electron transfer from the electron donor EDTA 4À to the excitedE Y 2À .  solvents. Very small shifts of their absorbance maximam ight result from Tyndall scattering of the liposomes suspension (Figure S8), while the shifti nl uminescence upon incorporation into liposomesw as hardly measurable ( % 2nm). Such minor spectroscopic variations suggest negligible solvent effects and minor aggregation of 1 2 + and 2 in phospholipid membranes as compared to organic solvent, whichdiffers from other oligovinylene chromophores. [25,36] Modeling the absorption spectra with time-dependent density functional theory (TD-DFT) yieldedt he loweste nergy absorptionb ands at 352 nm for 1 2 + + and 353 nm for 2 respectively,w hich is reasonably similart ot he experimental values ( Table 1). The CAMB3LYPf unctional wasc hosen for 1 2 + to take into account the charget ransfer (CT) character found for its lowest excited states:A ss hown in Figure 1d,t he calculated HOMO and LUMO of the ground state of 1 2 + + are located in the middle and at the extremities of the oligofluorene 1 2 + + ,r espectively.B yc ontrast, the HOMO and LUMO of 2 ( Figure S9) are both located at the center of the trifluorene molecule, lowest energy transition is amore classical p-p*character (Figure S9).
In order to see whether 1 2 + + aligns indeed perpendicularly to lipid membranes, confocal microscopy wasp erformed on giant multilamellar vesicles using laser excitation at 405 nm and detection in the region 420-514 nm ( Figure 1b). The luminescence images were superimposable with the simultaneously recorded transmission image ( Figure S16), which demonstrates that 1 2 + + is selectively taken up in the lipid bilayer.
For the reference compound 2 no selectives tainingo ft he bilayer was observed for 2 under comparablee xperimental conditions (see Figure S17), which we attribute to preferred pstacking of 2 over its solubility in the lipid bilayer structure.
Furthermore, for vesiclesw ith 1 2 + + ad ouble half-moon shaped emission profile was observed in all vesicles in the microscopici mage (Figure 1b), which is typical for molecules forming ac ircle in the observation plane. [25] The interaction of each chromophorem olecule with the laser beam depends on the orientationo ft heir transition dipole momentw ith respectt ot he direction of propagation of the light beam. As the incident laser light is polarized, all molecules with at ransition dipole moment (m T )p arallel to the polarization plane of the laser,a bsorb more light and therefore exhibit brighterl uminescence, which explainst he bright regions on the thick parts of both half-moons. In the thin regions of the image the transition dipole moment of 1 2 + + is orthogonal to the polarization plane,t herefore the absorption of the light beam, and hence the luminescence image are weaker.T he transition dipole momento ft he lowest electronic transition of 1 2 + + ,i sp arallel to the long axis of the molecule (Figure 1d)a nd has 6.32 Debyea ccordingt oT D-DFT calculation at the CAM-B3LYP/TZP level. Hence, spherically assembled transition dipole moments correspond to spherically assembled molecules.
In principle, one could argue that the half-moon effect might be due to either ap arallel, or ap erpendicular (transmembrane) alignment of 1 2 + + with respect to the lipid bilayer. We performed molecular dynamics simulations using Gromacs 2018 software [37] in order to check that. First, the self-assembly of 6independentrandom distributions of 128 DMPC molecules and one molecule of 1(PF 6 ) 2 in water was modelled for 200 ns, as described in the Supporting Information. In all cases spontaneous bilayer formation was observed, and in four cases out of six 1 2 + + indeed ended up in at ransmembrane fashion (sees upplementary movie Movie1.mpg), whereas two simulations ended up in ap arallel configuration. This result suggested a preference of 1 2 + + for at ransmembrane self-assembly,b ut it would not be affordable to quantify this preference using this computationally intensive method. Thus, in two of these simulationsw ec omputed the binding free energy of 1 2 + + to the membrane, DG bind either in the transmembrane or in the parallel configuration( see detailsi nt he Supporting Information). The averaged DG bind for the perpendicular (transmembrane) and parallel configurationw ere À165.5 kJ mol À1 and À22.4 kJ mol À1 ,r espectively,w hich further confirmed the preferenceo f1 2 + for the transmembrane configuration. Overall, these modeling studies supported our design hypothesis, that the half-mooneffect observed in confocal images of giant vesicles containing 1 2 + ,i sd ue to ap reference for at ransmembrane configuration of this linear molecule.
In nature,p hotosystem It ransfers the excitation energy of the transmembrane molecular light harvester to as econd dye in the membrane, to finally induce charget ransfer.T om imic this system eosin Y( EY 2À )w as chosen as ac o-dopanti nl ipid membranes,b ecause this dye has been widely used in photoelectron transfer [40] and photocatalytic protona nd CO 2 reduction studies on lipid bilayers and cell membranes. [22,40,41] Therefore, 1 2 + + and H 2 EY were added in different ratios into the lipid bilayer of DPPC liposomes during lipid film preparation. Deprotonation of H 2 EY to EY 2À occurred upon hydration of the lipid films with ap hosphate buffer at pH 7.8, as demonstrated by the characteristica bsorption maximum at 544 nm for DPPC:1 2 + + :EY 2À liposomes (1000:13:10 n/n/n ratio).I nterestingly,t his band is significantly red-shifted compared to homogeneous solution (l max = 517 nm in water [42][43][44] ). The absorbance of 1 2 + + was slightly blue-shifted in presence of EY 2À in the membrane, from 356 nm in DPPC:1 2 + + liposomes (1000:13n /n ratio) to 351 nm in DPPC:1 2 + + :EY 2À liposomes (1000:13:10 n/n/n ratio).B oth shifts are indicative of supramolecular interaction within the membrane between EY 2À and 1 2 + + (in the ground state). [42] These interactions were confirmed by molecular dynamics simulations of one molecule of 1 2 + + and one molecule of EY 2À in aD MPC lipid bilayer model.W ithin 30 ns simulation both dyes showedc lose contact interactions, characterized by ad istance of less than 1nmb etween the two oppositely charged species. Respectiveg raphical presentations of this modelc an be found in Figure S6 and Figure S7.
The formation of as upramolecular complex between 1 2 + + and EY 2À in liposomes was confirmed by efficient energy transfer from 1 2 + + to EY 2À observed upon selective photoexcitation of 1 2 + + (at 374 nm) lighting up the emission band of EY 2À (Figure 2b). The steady-state emission spectrumo fs uch DPPC:1 2 + + :EY 2À liposomes showedg radualq uenching of the emission of 1 2 + + at 404 nm upon adding increasing concentrationso fE Y 2À into the membrane,w hile increasinge mission of EY 2À was observed ( Figure 2b). Plottingt he inverse of the luminescence in-  Figure S15). Eq. (1) was used to obtain the association constant (K a in M À1 )f or the equilibrium shown in Eq. (2): [45] In Eq. (1), I 0 and I representt he emission intensity of 1 2 + + in absence and in presence of the quencher [EY 2À ], and K SV the Stern-Volmer constant (in M À1 )f or the dynamic quenching of the emissive S 1 excited state of 1 2 + + by EY 2À .I na bsence of EY 2À DPPC:1 2 + + liposomesh ad al uminescence lifetime of 1.4 ns. In the lower concentration regime of EY 2À ([EY 2À ] < 0.5 [1 2 + + ]) the dynamic quenching takes place with aS tern-Volmer constant K SV = 5.3·10 5 m À1 while the association constant( K a )f or its static component is K a = (2.1 AE 0.1) 10 6 m À1 .T his association constanti s3orders of magnitude stronger than the reported association of EY 2À to bare DPPC vesiclesa tp H7 (K a = (1.0 AE 0.1) 10 3 m À1 ) [39] whichh ighlights the strong attracting effect of the positivelyc harged membrane-doping agent 1 2 + + .A th igher concentration of EY 2À (0.5 < [EY 2À ]/[1 2 + + ] < 1) the quenching behavior does not follow the trend of eq. 1a nymore, which might be due to dimerization of EY 2À at the membrane interface. [46] Luminescenceq uenching was also observed by confocal luminescence microscopy of micrometer sized multi-lamellar giant vesicles.T he blue luminescence observed with DMPC vesicles containing 1 2 + + was quenched almost completely upon addition of 10 mm EY 2À to the outer aqueous phase of the giant vesicles, whilethe luminescence of EY 2À in the red region of the spectrum was switched on (Figure 2c). Interestingly, this phenomenon was not observed for apparently similar DPPC:1 2 + + vesicles. Upon addition of 10 mm EY 2À to the outer aqueous phase of thesev esicles at room temperature, the luminescence of 1 2 + + was only partly quenchedl ighting up only parts of the EY 2À luminescence. This could be explained by the fact that only the outer shells of the multi-lamellar vesicles are interacting with EY 2À .A ccording to the leakaget est with DPPC:1 2 + + (Supporting Information, p. S32), lipid bilayersa re impermeable to water-soluble species. Therefore, inner lamellas of multilamellar vesicles are not affectedb yq uenching via energy transfer.B yc ontrast, DMPC vesicles are inherently leaky and more fluid at room temperature, because their phase transition temperature coincides with room temperature. [47,48] Nevertheless, these data underline that the supramolecular complex [1 2 + + :EY 2À ]f orms within the phospholipid bilayer and provides an efficient scaffold for energy transfer from the transmembrane blue-lighth arvesting oligofluorene 1 2 + + to the photoredox catalystEY 2À .
To test the reactivity of the energy transferred on EY 2À for furtherr edox reactions, DPPC:1 2 + + :EY 2À liposomes (1000:13:10 n/n/n at 1mm DPPC) were irradiated at 375 nm (0.5 mW) in the presence of an isotonic buffer containing8 3mm EDTA 4À at pH 7.8. During irradiation the absorption band at 544 nm characteristic for EY 2À vanished with ar ate constant of 18 min À1 , while simultaneously the absorption band of 1 2 + + was shifted from 351 nm to 354 nm. (Figure 3a). Based on the excited state energies and redox potentials of all membrane-embedded components or their reference compound (Table2)t he reaction sequence shown in Scheme 1a nd Figure 3i sp roposed. Upon photoexcitation of 1 2 + + ,e nergy transfer (ET) takes place from an excited state of 1 2 + + to EY 2À .T his step has an overall driving force of 1.3 eV,e ither from the S 1 state of 1 2 + + at 3.2 eV to the S 1 state of EY 2À (2.3 eV) followed by intersystem crossing to the T 1 state of EY 2À at 1.9 eV, [40] or via inter system crossing of 1 2 + + to the T 1 state at 2.3 eV, [49] followed by triplet-triplet energy transfer to the triplet exciteds tate of EY 2À at 1.9 eV. [40] From its T 1 state EY 2À accepts an electron and two protons from the electron donor EDTA 4À with ad riving force DG eT = À0.2 eV,providing the almostcolorless EYH 2 2À . [50] The slow electron transfer kinetics on the minute time scale can be explained by the strong association of the relatively hydrophobic EY 2À dyes to the membrane,a ss upported by the stronga ssociation constant with 1 2 + and the close contact observed in molecular dynamics simulation (Supporting Info page S22-S23). By contrast, the strongly chargeda nd poorly hydrophobic species EDTA 4À is anticipated to remain in the aqueous phase. Still, the positive charge of the antenna 1 2 + + might playarole in attracting the anionic EDTA 4À electron donorn ear the membrane-water interface, therebyp romoting electron transfer from the excited state of EY 2À .A sa na lterna- tive, it may also be possible that in DPPC:1 2 + + :EY 2À liposomes EY 2À diffuses temporarily awayf rom the membrane into the solution,t oa bsorb photonsb yi tself and directlyp hotoreact with the sacrificial donor EDTA 4À in the aqueous phase, before stochastically coming backt othe membrane.
To investigate if the observed photoreduction may have occurred via direct photoexcitation of EY 2À by the 375 nm exciting light (0.1 10 4 m À1 cm À1 )a nd subsequentp hotoreduction by EDTA 4À ,werealized two control experiments. First, astrongly membrane-bound eosin Yd ye C16EY À was prepared by covalent functionalization of the acid side group with al ong (C16) aliphatic chain (Scheme 2). DPPC liposomes doped with 1mol %o fC 16EY À showed an absorption band similart oE Y 2À at pH 7.8 in water,b ut red-shifted to 545 nm. This is in line with the integration of the eosin dye into ah ydrophobic environment such as al ipid bilayer. [39,42] Irradiating DPPC:C16EY À liposomesw ith neither 375 nm nor 530 nm light in the presence of EDTA 4À (42 mm)d id not yield any spectroscopic changes.T herefore, no light-induced electron transfer occurred betweent he strongly membraneb ound excited state of C16EY À and EDTA 4À in the aqueous phase. Secondly,f ree eosin EY 2À (6.7 mm)w as quickly photoreduced in the presence of EDTA 4À (42 mm)i nh omogeneous, liposome-free buffer at pH 7.8 upon irradiation with 375 nm LED light (0.5 mW), as seen by the disappearance of the absorption band at 517 nm with ar ate constant of 1.15 AE 0.1 min À1 .T he evolution of the spectra is shown in Figure S19. This photoreaction rate is significantly faster than that observed with DPPC:C16EY À liposomes and DPPC:1 2 + + :EY 2À liposomes, whichi sm ost probably due to ac ombinationo fs everal effects. First, in absence of 1 2 + there is no filter effect by this strongly UV-absorbing molecule, so all availablel ight is absorbed by EY 2À and can lead to excited state formation. For DPPC:1 2 + + :EY 2À liposomes, 1 2 + + absorbs most light, preventing direct absorption by EY 2À .S econd, diffusion rates are higher in homogeneous solution than with molecules embedded in membranes,w hich may improve electron transfer rate in liposome-free conditions. Finally,i nD PPC:1 2 + + :EY 2À liposomes the strong association of EY 2À to 1 2 + + leads to av ery low bulk concentration of EY 2À in the water phase, which slows down directe lectron transfer from the excited states of EY 2À ,t oEDTA 4À .

Conclusions
Overall,o ur experimentala nd theoretical data are consistent with the following picture. First, the transmembrane oligofluorene 1 2 + + is acting as al ight-harvesting chromophore that selfassembles perpendicular to the membrane, andt ransfers photochemical energy to EY 2À within am embrane-embedded supramolecular complex. We proposet hat followinge nergy transfer,t he triplete xcited state of EY 2À is reduced at the membrane-wateri nterface by the reductant EDTA 4À ,t oac olorless form. To the best of our knowledge,t he combination of light absorption, energy transfer,a nd electron transfer using a transmembrane chromophore represents an unprecedented functional mimic of PS Iu sing simple organic chromophores.

Experimental Section
Experimental details including synthetic procedures can be found in the Supporting Information. Deposition Number 1970033 contains the supplementary crystallographic data for the structure of the brominated intermediate obtained during the synthesis of 1 2 + + .T hese data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.  3 %( 13 mm) 1 2 + + at 1mm DPPC and 10 mm EY 2À overall ratio of 1 2 + + /EY 2À is 1:0.8 (n/n) uponirradiation with3 75 nm LED light.Inset: Te mporal evolution of the absorbanceat 544 nm. b) Thermochemistry of energyt ransfer from photoe xcited 1 2 + + to EY 2À followed by electron transfer from exciteds tate EY 2À to the water-soluble electron acceptor EDTA 4À .