In situ time-resolved spectroelectrochemistry reveals limitations of biohybrid photoelectrode performance

•A protein-photoelectrode was investigated with spectroscopy and electrochemistry•We resolved electron flow and energy loss channels during biohybrid operation•Continuous intense irradiation exacerbated loss channels and compromised efficiency•The knowledge will help drive the rational design of more efficient biohybrids Solar energy fuels life in two ways: through photosynthesis, providing food for virtually all organisms on the Earth; and by generating electricity in photovoltaic devices. Biohybrid photoelectrodes combine the best of both worlds, coupling natural-light-driven photoenzymes with complex artificial materials in a bid to achieve sustainable, low-power tools. These may be used in a wide range of applications, from the production of solar fuels and value-added chemicals to electricity. However, bridging nature and technology often proves challenging, as biohybrid devices remain inefficient and unstable due to the creation of harmful side reactions. Using combined spectroscopic and electrochemical tools, we discover the origin of productivity losses during the operation of a state-of-the-art biohybrid device. A comprehensive quantification of loss processes is provided together with a blueprint for removal of these hurdles, bringing us closer to a zero-carbon economy driven by sustainable photoenzymes. Photosynthetic reaction centers catalyze the majority of solar energy conversion on the Earth. Under low-intensity illumination, this is achieved with a near-unity quantum efficiency, almost every absorbed photon producing a photochemical charge separation. Biohybrid technologies seek to capture the high efficiency of natural photoproteins by combining them with man-made electrodes. However, the transfer of photoproteins from their membrane environment into an abiotic architecture invariably results in efficiency losses. Here, we combined spectroscopy and analytical electrochemistry to identify the loss processes in a reaction-center-based biophotoelectrode. While over 90% efficient under low-intensity illumination, the biophotoelectrode efficiency dropped to ∼11% under high-intensity illumination. This loss stemmed from bottlenecks in electron transfer that rendered 60% of reaction centers inactive, as well as a short-circuiting of 73% of the separated charge from active reaction centers. The quantitative insights into loss processes presented in this work will be instrumental in shaping future rational design of biophotoelectrode devices. Photosynthetic reaction centers catalyze the majority of solar energy conversion on the Earth. Under low-intensity illumination, this is achieved with a near-unity quantum efficiency, almost every absorbed photon producing a photochemical charge separation. Biohybrid technologies seek to capture the high efficiency of natural photoproteins by combining them with man-made electrodes. However, the transfer of photoproteins from their membrane environment into an abiotic architecture invariably results in efficiency losses. Here, we combined spectroscopy and analytical electrochemistry to identify the loss processes in a reaction-center-based biophotoelectrode. While over 90% efficient under low-intensity illumination, the biophotoelectrode efficiency dropped to ∼11% under high-intensity illumination. This loss stemmed from bottlenecks in electron transfer that rendered 60% of reaction centers inactive, as well as a short-circuiting of 73% of the separated charge from active reaction centers. The quantitative insights into loss processes presented in this work will be instrumental in shaping future rational design of biophotoelectrode devices. Many man-made devices for solar energy conversion operate by mechanisms that have similarities to, or are inspired by, those that take place in the photosystems of plants, algae, and bacteria. A much remarked upon feature of the primary reactions of natural photosynthesis, energy transfer within light-harvesting pigment-proteins and charge separation in photochemical reaction centers (RCs), is their high quantum efficiency (events per photon absorbed) that approaches 100% in yield at low light intensity.1Croce R. van Amerongen H. Natural strategies for photosynthetic light harvesting.Nat. Chem. Biol. 2014; 10: 492-501https://doi.org/10.1038/nchembio.1555Crossref PubMed Scopus (639) Google Scholar This innate efficiency has driven interest in the direct utilization of natural and engineered pigment-protein complexes in biohybrid devices.2Sokol K.P. Mersch D. Hartmann V. Zhang J.Z. Nowaczyk M.M. Rögner M. Ruff A. Schuhmann W. Plumeré N. Reisner E. 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In the case of the reaction center light-harvesting 1 (RC-LH1) complex from the anoxygenic photosynthetic bacterium Rhodobacter (Rba.) sphaeroides, its location within an architecture provided by the photosynthetic lipid bilayer membrane prevents electrical short circuits between mobile carriers of charge (Figure 1A). Accordingly, removal of the RC-LH1 complex from the lipid bilayer for interfacing with electrodes may introduce loss processes such as short circuits (process 4 in Figure 1C).5van Rotterdam B.J. Westerhoff H.V. Visschers R.W. Jones M.R. Hellingwerf K.J. Crielaard W. Steady-state cyclic electron transfer through solubilized Rhodobacter sphaeroides reaction centres.Biophys. 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Sci. 2022; 23: 4774https://doi.org/10.3390/ijms23094774Crossref PubMed Scopus (4) Google Scholar Furthermore, no study to date has provided a comprehensive method that empirically deconvolutes all electron transfer pathways and loss processes in a biophotoelectrode exposed to continuous illumination and under operating conditions of an applied potential. Investigations in situ and in operandum are crucial for the rational design of high-performing biohybrids, particularly in the context of recent and more complex benchmark architectures that drive large photocurrent densities but encounter multiple loss channels from short circuits, electron transfer bottlenecks, and excitation quenching.4Friebe V.M. Barszcz A.J. Jones M.R. Frese R.N. Sustaining electron transfer pathways extends biohybrid photoelectrode stability to years.Angew. Chem. Int. Ed. Engl. 2022; 61: e202201148https://doi.org/10.1002/anie.202201148Crossref PubMed Scopus (4) Google Scholar,22Friebe V.M. Delgado J.D. 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Delacotte J. Rappaport F. Wollman F.A. Guille-Collignon M. Lemaître F. Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time.Chem. Sci. 2018; 9: 8271-8281https://doi.org/10.1039/c8sc03058hCrossref PubMed Scopus (38) Google Scholar In this work, we utilized a combined spectroelectrochemical instrument, based on a ms/μs pump-probe Joliot-type spectrophotometer, to measure the redox states of Rba sphaeroides RC-LH1 complexes adsorbed on a semi-transparent mesoporous indium tin oxide electrode (mITO - Figure 1D). By simultaneously recording photocurrents and spectrally derived functional bRC oxidation states, kinetic bottlenecks and loss processes were identified at various stages of excitation and electron transfer in this mITO|cyt c|RC-LH1 electrode. We found that 73% of energy loss stemmed from short-circuiting between the product of photochemical charge separation and the electrode, as well as 60% of bRCs that were not capable of performing photochemistry because of donor- and acceptor-side bottlenecks. We further extended this approach to accommodate a range of case studies to demonstrate the capabilities of our method to pinpoint various bottlenecks and loss processes. Furthermore, the approach can be adapted to systems that utilize other photoproteins such as photosystems I and II, to aid the rational design of biohybrid electrodes at each step of their electron and excitation energy transfer pathways. The comprehensive overview and understanding of these loss processes derived with our spectroelectrochemical approach will be instrumental in harnessing the high catalytic efficiency of natural photoproteins in biohybrid devices. To simultaneously investigate photocurrent generation through electrochemistry and spectroscopy a custom spectroelectrochemical cell was constructed and placed in the measuring light beam of a ms/μs pump-probe spectrophotometer (Figure 2A, detailed side view in Figure S2B). The cell was fitted with an mITO|cyt c|RC-LH1 electrode that was immersed in a buffer containing 5 mM ubiquinone (Q0) and 50 mM KCl as electrolyte. The cell also contained reference and counter electrodes connected to a potentiostat that controlled the ambient redox potential used to fix the redox state of donors and acceptors. An applied potential of +160 mV vs. SHE was used to optimally drive a photocathodic current, as described previously for cyt c mediated bRC photocathodes.4Friebe V.M. Barszcz A.J. Jones M.R. Frese R.N. Sustaining electron transfer pathways extends biohybrid photoelectrode stability to years.Angew. Chem. Int. Ed. Engl. 2022; 61: e202201148https://doi.org/10.1002/anie.202201148Crossref PubMed Scopus (4) Google Scholar,26Friebe V.M. Frese R.N. Photosynthetic reaction center-based biophotovoltaics.Curr. Opin. Electrochem. 2017; 5: 126-134https://doi.org/10.1016/j.coelec.2017.08.001Crossref Scopus (51) Google Scholar Excitation was provided either by an LED peaking at 590 nm, or by 5 ns laser flashes at 532 nm (Figures 2A and 2B). Photooxidation of the bRC primary electron donor was monitored at 865 nm (ϵ865 = 112 ± 6 mM−1 cm−1, see Straley et al.27Straley S.C. Parson W.W. Mauzerall D.C. Clayton R.K. Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides.Biochim. Biophys. Acta. 1973; 305: 597-609https://doi.org/10.1016/0005-2728(73)90079-0Crossref PubMed Scopus (268) Google Scholar), with simultaneous recording of photoinduced electron transfer at the electrode interface using chronoamperometry. Both processes were initially studied using saturating laser flashes that were sufficiently brief that they induced no more than a single charge separation per bRC (“single-turnover conditions”), but sufficiently intense to trigger photochemistry in all bRCs. As with chemical oxidation (Figure 2B), laser flashes induced a characteristic broad bleach from 820 to 950 nm corresponding to the formation of P870+ (see Białek et al.28Białek R. Friebe V. Ruff A. Jones M.R. Frese R. Gibasiewicz K. In situ spectroelectrochemical investigation of a biophotoelectrode based on photoreaction centers embedded in a redox hydrogel.Electrochim. Acta. 2020; 330https://doi.org/10.1016/j.electacta.2019.135190Crossref Scopus (9) Google Scholar), which decayed on a μs-s time scale (Figure 3A). As P870 can be (electro)chemically oxidized, its full reduction before the excitation flash was ensured by applying a potential of +160 mV vs. SHE. At such a potential bRCs also have oxidized quinone electron acceptors (Figure 1E) and are in the open state required to perform a productive charge separation. Three independent approaches were used to confirm that 100% of bRCs were open under these conditions: single-turnover flashes, continuous LED illumination in the absence of cyt c, and continuous illumination at a more positive applied potential (Figure S3). All methods produced the same maximal amplitude of the absorbance change at 865 nm, diagnostic of P870+. Under conditions with oxidized QA and QB quinone acceptors, post-flash recovery of P870 on the μs-s time scale (Figure 3A) is due to a competition between reduction of P870+ by cyt c and recombination of the radical pair P870+QA− or P870+QB− (simplified to P870+Q− below). Consequently, by varying the concentration of free cyt c, it was possible to resolve multiple kinetic phases of P870+ reduction (see Figure S4 and associated supplementary text). Complementary to transient absorption measurements, photocurrents induced by single-turnover flash excitation were simultaneously monitored. Upon light-induced P870+ formation, cyt c2+ will be oxidized to cyt c3+, undock from the bRC, and transport the electron-hole to the electrode, causing a burst of photocurrent (Jphoto) with an onset at around 20 ms post-flash (Figure 3A, green). Integration of the transient photocurrent density and conversion with Faraday’s constant revealed the cumulative amount of cyt c oxidized during the burst of Jphoto to be of the order of 100 pmol cyt c cm−2 (Figure 3A, cyan). This correlated well with an estimate of 95 pmol cm−2 for the quantity of RC-LH1 complex adsorbed on the electrode, determined by pigment extraction (see experimental procedures). This indicated that, upon a single photochemical event per bRC, practically all electron-holes generated on P870 were transferred to the electrode, producing a near-unity quantum efficiency (compare the right axes in Figure 3A). The high efficiency was maintained over a train of 20 flashes, leading to equal cumulative amounts of oxidized P870+ and cyt c3+ (Figure 3B, blue and orange). This indicated that electron transfer in these conditions, with a low effective light intensity producing one charge separation s−1 bRC−1, reproducibly proceeded with near quantum unity efficiency over multiple turnovers. While the single-turnover flash method is informative in probing the kinetics of interfacial electron transfer and its maximal efficiency, benchmark operating conditions for solar cell devices comprise continuous 1,000 W cm−2 photon fluxes.29Gueymard C.A. Myers D. Emery K. Proposed reference irradiance spectra for solar energy systems testing.Sol. Energy. 2002; 73: 443-467https://doi.org/10.1016/S0038-092X(03)00005-7Crossref Scopus (444) Google Scholar This irradiance intensity is approximately 3,500 μmol photons m−2 s−1 in the photosynthetically active radiation region of RC-LH1 from 400 to 900 nm. Furthermore, while the redox states of all mediators equilibrate with the applied potential in the dark period between single-turnover flashes, redox states shift significantly under continuous turnover conditions. The performance and redox state of the electrode-bound RC-LH1 complex was therefore also examined under continuous illumination. To partition bRCs between open, oxidized and closed states during continuous illumination, absorption changes corresponding to P870 oxidation were used. As described above (see Figure S3), the maximal extent of P870+ in situ was obtained with three independent approaches. This amplitude could then be compared with the amount of P870 oxidized upon actinic light illumination to obtain the fraction of oxidized bRCs; and by employing multi-turnover, saturating light pulses, the remaining fraction of reduced bRCs can be separated into open and closed states. The fraction of oxidized bRCs was monitored under conditions optimal for photocurrents (+160 mV vs. SHE), an applied potential at which the cyt c donor is reduced and quinone acceptors are oxidized. The observed absorbance change indicated that ∼40% of bRCs became oxidized within 250 ms (Figure 4B, black trace and inset). The transient increase in P870+ upon each multi-turnover pulse reports on the proportion of open bRCs that are able to form a metastable P870+/QB− state (or P870+/QBH2 given that the QB quinone is a two-electron carrier) and is shaded in green in Figure 4B. This fraction of open bRCs stabilized at ∼32% over the 40-s illumination period. The accumulation of these oxidized bRCs (bearing P870+) under actinic light illumination depicts the donor-side limitation under operating conditions. The fraction of bRCs which were open (i.e., ready to perform stable photochemistry, Figure 4B) was assessed by probing with additional saturating pulses. The remaining fraction, colored red in Figure 4B, constituted the population of closed bRCs that cannot form a metastable P870+ state due to an acceptor-side limitation. This limitation, due to a long-lived QA− present in the closed centers, induces an ultrafast recombination between P870+ and the bacteriopheophytin anion (HA−) (Figure S1G). Over the illumination period the population of closed bRCs grew, shifting the fraction of bottlenecked bRCs from predominantly donor-side limited (at around 1 s of illumination) to roughly equal parts donor- and acceptor-side limited under steady-state conditions (Figure 4B). To get a better grasp on the molecular photochemical events taking place on the mITO|cyt c|RC-LH1 electrode, we proceeded to quantify the system in terms of electron transfer rates (ETR) on a “per bRC” basis. Under continuous illumination the steady-state ETR is a product of the fraction of open bRCs (φRC) and their maximal, light-limited charge separation rate (kcs; combining the average time between photon absorption, excitation energy transfer to the bRC, and stable charge separation):ETR=φRC∗kcs(Equation1) To determine kcs, the initial rise in P870+ formation upon illumination under an applied potential of +360 mV vs. SHE was fitted with a single exponential (Figure 4A, inset). This allowed determination of kcs in the absence of P870+ re-reduction, a methodology analogous to a recent examination of PSI in the absence of its electron donor, plastocyanin.30Nawrocki W.J. Santabarbara S. Mosebach L. Wollman F.A. Rappaport F. State transitions redistribute rather than dissipate energy between the two photosystems in Chlamydomonas.Nat. Plants. 2016; 2: 16031https://doi.org/10.1038/nplants.2016.31Crossref PubMed Google Scholar A value of 110 electrons s−1 bRC−1 was obtained for kcs at the 2,600 μmol photons m−2 s−1 irradiance, in reasonable agreement with a theoretical calculated value of 186 electrons s−1 RC-LH1−1 based on the light spectrum, the absorption spectrum of RC-LH1 complexes, and the geometry of the cuvette (Figure S5). Scattering from the electrode and shading was not taken into account, which may explain the remaining discrepancy. Multiplication of the fraction of open bRCs capable of performing photochemistry (φRC in Figure 4B) by kcs produced an average ETR per-bRC during operation (Figure 4D, blue line). This spectroscopy-derived ETR stabilized at just under 44 electrons s−1 bRC−1 after 40 s of illumination under these conditions. In conclusion, under these steady-state conditions only 40% of the bRCs are capable of photochemistry. The evolution of bRC ETR during the illumination window was also derived from electrochemical data by converting photocurrent densities (Figure 4C) into values of apparent ETR as described in the experimental procedures. It is crucial to note that such electrochemically derived values of ETR, often called turnover frequency (TOF), are apparent values since electrochemistry only probes the interfacial electron flux with cyt c and QH2 (Figure 4E). In contrast, the spectroscopic data accurately reflect turnover at the RC-LH1 complex and include contributions from short-circuiting, quenching, or leaks that cannot otherwise be extracted electrochemically. Strikingly, values for the spectroscopy-based ETR exceeded those of ETRapp derived from electrochemical measurements. We interpret this discrepancy as a methodological feat that allows the extent of short circuits present in the biohybrid electrode to be revealed. In contrast to the spectroscopy derived 44 e− s−1 bRC−1 ETR, the electrochemistry-based ETR stabilized at 12 e− s−1 bRC−1 at the end of the 40 s illumination window (Figure 4D). This indicates that ∼73% of the ETR resulted in non-productive photochemistry under steady-state conditions, primarily due to short circuits. Because short circuits can be of various types (see Figure S1C), we propose that the kinetic behavior of the signal allows at least two subtypes to be distinguished. As illustrated in Figure 4E, two expected short circuits involving reduced quinone are electron donation to the electrode (SC2) or to cyt c3+ and/or P870+ (SC1). As validated by the loss of photocurrent decline on stirring the electrolyte and by the presence of inverted Jphoto following the light-to-dark transition,22Friebe V.M. Delgado J.D. Swainsbury D.J.K. Gruber J.M. Chanaewa A. Van Grondelle R. Von Hauff E. Millo D. Jones M.R. Frese R.N. Plasmon-enhanced photocurrent of photosynthetic pigment proteins on nanoporous silver.Adv. Funct. Mater. 2016; 26: 285-292https://doi.org/10.1002/adfm.201504020Crossref Scopus (84) Google Scholar SC2 accounts for the increasing variance between the spectroscopy-based ETR and electrochemistry-based ETR during the illumination period (Figure 4D). The magnitude of SC2 was extrapolated from the reverse current spike (Figure S6A), and the remaining losses were attributed to SC1. Considering the magnitude of SC1 was relatively small, SC2 was attributed as the major short-circuiting pathway. A range of case studies were performed to illustrate the versatility of spectroelectrochemistry to pinpoint and overcome bottlenecks and loss processes under various operating conditions. Lowering the quinone concentration from 5 to 1 mM resulted in clear acceptor-side limitations in