ABSTRACT
Solar fuels catalysis is a promising route to efficiently harvesting, storing, and utilizing abundant solar energy. To achieve this promise, however, molecular systems must be designed with sustainable components that can balance numerous photophysical and chemical processes. To that end, we report on the structural and photophysical characterization of a series of Cu(I)-anthraquinone-based electron donor-acceptor dyads. The dyads utilized a heteroleptic Cu(I) bis-diimine architecture with a copper(I) bis-phenanthroline chromophore donor and anthraquinone electron acceptor. We characterized the structures of the complexes using x-ray crystallography and density functional theory calculations and the photophysical properties via resonance Raman and optical transient absorption spectroscopy. The calculations and resonance Raman spectroscopy revealed that excitation of the Cu(I) metal-to-ligand charge-transfer (MLCT) transition transfers the electron to a delocalized ligand orbital. The optical transient absorption spectroscopy demonstrated that each dyad formed the oxidized copper-reduced anthraquinone charge-separated state. Unlike most Cu(I) bis-phenanthroline complexes where increasingly bulky substituents on the phenanthroline ligands lead to longer MLCT excited-state lifetimes, here, we observe a decrease in the long-lived charge-separated state lifetime with increasing steric bulk. The charge-separated state lifetimes were best explained in the context of electron-transfer theory rather than with the energy gap law, which is typical for MLCT excited states, despite the complete conjugation between the phenanthroline and anthraquinone moieties.
ABSTRACT
A nitrogen K-edge x-ray absorption near-edge structure (XANES) survey is presented for tetrapyrido[3,2-a:2',3'-c:3â³,2â³-h:2â´,3â´-j]phenazine (tpphz)-bridged bimetallic assemblies that couple chromophore and catalyst transition metal complexes for light driven catalysis, as well as their individual molecular constituents. We demonstrate the high N site sensitivity of the N pre-edge XANES features, which are energetically well-separated for the phenazine bridge N atoms and for the individual metal-bound N atoms of the inner coordination sphere ligands. By comparison with the time-dependent density functional theory calculated spectra, we determine the origins of these distinguishable spectral features. We find that metal coordination generates large shifts toward higher energy for the metal-bound N atoms, with increasing shift for 3d < 4d < 5d metal bonding. This is attributed to increasing ligand-to-metal σ donation that increases the effective charge of the bound N atoms and stabilizes the N 1s core electrons. In contrast, the phenazine bridge N pre-edge peak is found at a lower energy due to stabilization of the low energy electron accepting orbital localized on the phenazine motif. While no sensitivity to ground state electronic coupling between the individual molecular subunits was observed, the spectra are sensitive to structural distortions of the tpphz bridge. These results demonstrate N K-edge XANES as a local probe of electronic structure in large bridging ligand motifs, able to distinctly investigate the ligand-centered orbitals involved in metal-to-ligand and ligand-to-ligand electron transfer following light absorption.
ABSTRACT
A heteroleptic copper(I) bis(phenanthroline) complex with aldehyde groups at the 4,7 positions of the phenanthroline ligand was synthesized. The complex is responsive to alcohol, resulting in a distinct colour change caused by the facile reaction of the aldehyde group with alcohol, forming a hemiacetal product. The aldehyde species can be regenerated after heating the intermediate at 80 °C for 10 minutes, demonstrating the reusability of the complex for alcohol detection. This work presents a new strategy for applying transition metal complexes in small molecule sensing by installing functional groups in the secondary coordination sphere which reversibly react with analytes.
ABSTRACT
A key challenge to the effective utilization of solar energy is to promote efficient photoinduced charge transfer, specifically avoiding unproductive, circuitous electron-transfer pathways and optimizing the kinetics of charge separation and recombination. We hypothesize that one way to address this challenge is to develop a fundamental understanding of how to initiate and control directional photoinduced charge transfer, particularly for earth-abundant first-row transition-metal coordination complexes, which typically suffer from relatively short excited-state lifetimes. Here, we report a series of functionalized heteroleptic copper(I)bis(phenanthroline) complexes, which have allowed us to investigate the directionality of intramolecular photoinduced metal-to-ligand charge transfer (MLCT) as a function of the substituent Hammett parameter. Ultrafast transient absorption suggests a complicated interplay of MLCT localization and solvent interaction with the Cu(II) center of the MLCT state. This work provides a set of design principles for directional charge transfer in earth-abundant complexes and can be used to efficiently design pathways for connecting the molecular modules to catalysts or electrodes and integration into systems for light-driven catalysis.
ABSTRACT
The structure, chemistry, and charge of interfaces between materials and aqueous fluids play a central role in determining properties and performance of numerous water systems. Sensors, membranes, sorbents, and heterogeneous catalysts almost uniformly rely on specific interactions between their surfaces and components dissolved or suspended in the water-and often the water molecules themselves-to detect and mitigate contaminants. Deleterious processes in these systems such as fouling, scaling (inorganic deposits), and corrosion are also governed by interfacial phenomena. Despite the importance of these interfaces, much remains to be learned about their multiscale interactions. Developing a deeper understanding of the molecular- and mesoscale phenomena at water/solid interfaces will be essential to driving innovation to address grand challenges in supplying sufficient fit-for-purpose water in the future. In this Review, we examine the current state of knowledge surrounding adsorption, reactivity, and transport in several key classes of water/solid interfaces, drawing on a synergistic combination of theory, simulation, and experiments, and provide an outlook for prioritizing strategic research directions.
ABSTRACT
Facilitating photoinduced electron transfer (PET) while minimizing rapid charge-recombination processes to produce a long-lived charge-separated (CS) state represents a primary challenge associated with achieving efficient solar fuel production. Natural photosynthetic systems employ intermolecular interactions to arrange the electron-transfer relay in reaction centers and promote a directional flow of electrons. This work explores a similar tactic through the synthesis and ground- and excited-state characterization of two Cu(I)bis(phenanthroline) chromophores with homoleptic and heteroleptic coordination geometries and which are functionalized with negatively charged sulfonate groups. The addition of sulfonate groups enables solubility in pure water, and it also induces assembly with the dicationic electron acceptor methyl viologen (MV2+) via bimolecular, dynamic electrostatic interactions. The effect of the sulfonate groups on the ground- and excited-state properties was evaluated by comparison with the unsulfonated analogues in 1:1 acetonitrile/water. The excited-state lifetimes for all sulfonated complexes are similar to what we expect from previous literature, with the exception of the sulfonated heteroleptic complex whose metal-to-ligand charge-transfer (MLCT) lifetime in water has two components that are fit to 10 and 77 ns. For the sulfonated complexes, we detected reduced MV+⢠in both solvent environments following MLCT excitation, but control measurements in 1:1 acetonitrile/water with the unsulfonated analogues showed no PET to MV2+, indicating that electrostatically driven supramolecular assemblies of the sulfonated complexes with MV2+ facilitate the observed PET. Additionally, the strength of the intermolecular interactions driving the formation of these assemblies changes drastically with the solvent environment. In 1:1 acetonitrile/water, PET occurred from both sulfonated complexes with quantum yields (ΦET) of 2-3% but increased to a remarkable 98% for the sulfonated heteroleptic complex with a 3 µs CS-state lifetime in water.
Subject(s)
Phenanthrolines , Water , Ligands , Solvents , AcetonitrilesABSTRACT
High-energy X-ray scattering and pair distribution function analysis (HEXS/PDF) is a powerful method to reveal the structure of materials lacking long-range order, but is underutilized for molecular complexes in solution. We demonstrate the application of HEXS/PDF with 0.26â Å resolution to uncover the solution structure of five bimetallic CuI /RuII /OsII complexes. HEXS/PDF of each complex in acetonitrile solution confirms the pairwise distances in the local coordination sphere of each metal center as well as the metalâ â â metal distances separated by over 12â Å. The metalâ â â metal distance detected in solution is compared with that from the crystal structure and molecular models to confirm that distortions to the metal bridging ligand are unique to the solid state. This work presents the first example of observing sub-Ångström conformational differences by direct comparison of solution phase and solid-state structures and shows the potential for HEXS/PDF in the determination of solution structure of single molecules.
ABSTRACT
To understand design principles for assembling photosynthetic biohybrids that incorporate precisely-controlled sites for electron injection into redox enzyme cofactor arrays, we investigated the influence of chirality in assembly of the photosensitizer ruthenium(II)bis(2,2'-bipyridine)(4-bromomethyl-4'-methyl-2,2'-bipyridine), Ru(bpy)2(Br-bpy), when covalently conjugated to cysteine residues introduced by site-directed mutagenesis in the triheme periplasmic cytochrome A (PpcA) as a model biohybrid system. For two investigated conjugates that show ultrafast electron transfer, A23C-Ru and K29C-Ru, analysis by circular dichroism spectroscopy, CD, demonstrated site-specific chiral discrimination as a factor emerging from the close association between [Ru(bpy)3]2+ and heme cofactors. CD analysis showed the A23C-Ru and K29C-Ru conjugates to have distinct, but opposite, stereoselectivity for the Λ and Δ-Ru(bpy)2(Br-bpy) enantiomers, with enantiomeric excesses of 33.1% and 65.6%, respectively. In contrast, Ru(bpy)2(Br-bpy) conjugation to a protein site with high flexibility, represented by the E39C-Ru construct, exhibited a nearly negligible chiral selectivity, measured by an enantiomeric excess of 4.2% for the Λ enantiomer. Molecular dynamics simulations showed that site-specific stereoselectivity reflects steric constraints at the conjugating sites and that a high degree of chiral selectivity correlates to reduced structural disorder for [Ru(bpy)3]2+ in the linked assembly. This work identifies chiral discrimination as means to achieve site-specific, precise geometric positioning of introduced photosensitizers relative to the heme cofactors in manner that mimics the tuning of cofactors in photosynthesis.
Subject(s)
Biomimetics , Coordination Complexes/chemistry , Hemeproteins/metabolism , Photosensitizing Agents/chemistry , Photosynthesis , Circular Dichroism , Cysteine/genetics , Molecular Dynamics Simulation , Mutant Proteins/chemistry , Mutation/genetics , Spectrum Analysis , StereoisomerismABSTRACT
Worldwide there is a large research investment in developing solar fuel systems as clean and sustainable sources of energy. The fundamental mechanisms of natural photosynthesis can provide a source of inspiration for these studies. Photosynthetic reaction center (RC) proteins capture and convert light energy into chemical energy that is ultimately used to drive oxygenic water-splitting and carbon fixation. For the light energy to be used, the RC communicates with other donor/acceptor components via a sophisticated electron transfer scheme that includes electron transfer reactions between soluble and membrane bound proteins. Herein, we reengineer an inherent interprotein electron transfer pathway in a natural photosynthetic system to make it photocatalytic for aqueous H2 production. The native electron shuttle protein ferredoxin (Fd) is used as a scaffold for binding of a ruthenium photosensitizer and H2 catalytic function is imparted to its partner protein, ferredoxin-NADP+-reductase (FNR), by attachment of cobaloxime molecules. We find that this 2-protein biohybrid system produces H2 in aqueous solutions via light-induced interprotein electron transfer reactions (TON > 2500 H2/FNR), providing insight about using native protein-protein interactions as a method for fuel generation.
Subject(s)
Hydrogen/metabolism , Light , Anabaena/enzymology , Catalysis/radiation effects , Catalytic Domain , Electron Transport/radiation effects , Ferredoxin-NADP Reductase/chemistry , Ferredoxin-NADP Reductase/metabolism , NADP/metabolism , Osmolar Concentration , Photosensitizing Agents/chemistry , Ruthenium/chemistry , Time FactorsABSTRACT
Two new Co(II) complexes have been synthesized and investigated as catalysts for H2 generation. These catalysts were designed to incorporate redox-active bipyridine components and nitrogen groups, which can participate in electron and proton transfer steps in the catalytic cycle. The two catalysts differ by only one amino group, yielding a completely closed macrocycle and an open "macrocycle" complex. Removing just one nitrogen linker between the Co(II)-binding bipyridine groups has a profound impact on the molecular geometry observed by single crystal analysis. Photocatalysis experiments show that both catalysts are highly active for aqueous proton reduction at moderate pH levels, with the closed macrocycle reaching almost 2 × 104 turnovers of H2 when photodriven by [Ru(2,2'-bipyridine)3]2+ using ascorbate as an electron relay and a phosphine compound as the terminal electron donor. Measurements of the electrocatalytic activity were used to investigate key steps in the mechanism of proton reduction by the molecular catalysts. The formation of a new reversible peak on addition of moderately strong acids in organic solvents suggests that protonation of the macrocycle plays an important role in H2 generation. Onset of the catalytic current occurs near the reduction potential of the bipyridine components, suggesting that catalysis is mediated by electron transfer from the macrocycle to the cobalt center. From these observations, we propose a mechanism for catalytic proton reduction to H2, which involves both intramolecular proton and electron transfer steps from the macrocycle ligand to the cobalt center. The vital role of the second coordination sphere in the catalytic cycle places these relatively simple complexes on the pathway toward molecular catalysts that mimic the valuable features of enzymatic catalysis.
ABSTRACT
Photosynthetic reaction center (RC) proteins convert incident solar energy to chemical energy through a network of molecular cofactors which have been evolutionarily tuned to couple efficient light-harvesting, directional electron transfer, and long-lived charge separation with secondary reaction sequences. These molecular cofactors are embedded within a complex protein environment which precisely positions each cofactor in optimal geometries along efficient electron transfer pathways with localized protein environments facilitating sequential and accumulative charge transfer. By contrast, it is difficult to approach a similar level of structural complexity in synthetic architectures for solar energy conversion. However, by using appropriate self-assembly strategies, we anticipate that molecular modules, which are independently synthesized and optimized for either light-harvesting or redox catalysis, can be organized into spatial arrangements that functionally mimic natural photosynthesis. In this Account, we describe a modular approach to new structural designs for artificial photosynthesis which is largely inspired by photosynthetic RC proteins. We focus on recent work from our lab which uses molecular modules for light-harvesting or proton reduction catalysis in different coordination geometries and different platforms, spanning from discrete supramolecular assemblies to molecule-nanoparticle hybrids to protein-based biohybrids. Molecular modules are particularly amenable to high-resolution characterization of the ground and excited state of each module using a variety of physical techniques; such spectroscopic interrogation helps our understanding of primary artificial photosynthetic mechanisms. In particular, we discuss the use of transient optical spectroscopy, EPR, and X-ray scattering techniques to elucidate dynamic structural behavior and light-induced kinetics and the impact on photocatalytic mechanism. Two different coordination geometries of supramolecular photocatalyst based on the [Ru(bpy)3](2+) (bpy = 2,2'-bipyridine) light-harvesting module with cobaloxime-based catalyst module are compared, with progress in stabilizing photoinduced charge separation identified. These same modules embedded in the small electron transfer protein ferredoxin exhibit much longer charge-separation, enabled by stepwise electron transfer through the native [2Fe-2S] cofactor. We anticipate that the use of interchangeable, molecular modules which can interact in different coordination geometries or within entirely different structural platforms will provide important fundamental insights into the effect of environment on parameters such as electron transfer and charge separation, and ultimately drive more efficient designs for artificial photosynthesis.
Subject(s)
Coordination Complexes/chemistry , Photosynthesis , Photosynthetic Reaction Center Complex Proteins/chemistry , Catalysis , Cobalt , Ferredoxins/chemistry , Light , Nanoparticles/chemistry , RubidiumABSTRACT
This tutorial review illustrates opportunities for the resolution of structure-function relationships to aid in the development of new materials for solar energy conversion using a combination of spectroscopy and catalysis measurements with X-ray scattering analyses to provide in situ structural characterization of solar fuels catalysts. As an example, the use of molecular cobaloxime catalysts in bimolecular and supramolecular photocatalysis schemes for proton reduction is briefly reviewed. These highlight the need to develop new modular, hierarchical, self-healing supramolecular architectures for solar fuels catalysis. Examples of the X-ray scattering structural analysis of amorphous materials in the context of photocatalytic function are discussed in detail.
ABSTRACT
Photosynthetic reaction center (RC) proteins are finely tuned molecular systems optimized for solar energy conversion. RCs effectively capture and convert sunlight with near unity quantum efficiency utilizing light-induced directional electron transfer through a series of molecular cofactors embedded within the protein core to generate a long-lived charge separated state with a useable electrochemical potential. Of current interest are new strategies that couple RC chemistry to the direct synthesis of energy-rich compounds. This Feature Article highlights recent work from our lab on RC and RC-inspired hybrid systems that capture the Sun's energy and convert it to chemical energy in the form of H2, a carbon-neutral energy source derived from water. Biohybrids made from the Photosystem I (PSI) RC are among the best photocatalytic H2-producing protein hybrids to date. Targeted self-assembly strategies that couple abiotic catalysts to PSI translate to catalyst incorporation at intrinsic PSI sites within thylakoid membranes to achieve complete solar water-splitting systems. RC-inspired biohybrids interface synthetic photosensitizers and molecular catalysts with small proteins to create photocatalytic systems and enable the spectroscopic discernment of the structural features and electron transfer processes that underpin solar-driven proton reduction. In total, these studies showcase the incredible scientific opportunities photosynthetic biohybrid research provides for harnessing the optimal qualities of both artificial and natural photosynthetic systems and developing materials that capture, convert, and store solar energy as a fuel.
ABSTRACT
Appreciating that the role of the solute-solvent and other outer-sphere interactions is essential for understanding chemistry and chemical dynamics in solution, experimental approaches are needed to address the structural consequences of these interactions, complementing condensed-matter simulations and coarse-grained theories. High-energy X-ray scattering (HEXS) combined with pair distribution function analysis presents the opportunity to probe these structures directly and to develop quantitative, atomistic models of molecular systems in situ in the solution phase. However, at concentrations relevant to solution-phase chemistry, the total scattering signal is dominated by the bulk solvent, prompting researchers to adopt a differential approach to eliminate this unwanted background. Though similar approaches are well established in quantitative structural studies of macromolecules in solution by small- and wide-angle X-ray scattering (SAXS/WAXS), analogous studies in the HEXS regime-where sub-ångström spatial resolution is achieved-remain underdeveloped, in part due to the lack of a rigorous theoretical description of the experiment. To address this, herein we develop a framework for differential solution scattering experiments conducted at high energies, which includes concepts of the solvent-excluded volume introduced to describe SAXS/WAXS data, as well as concepts from the time-resolved X-ray scattering community. Our theory is supported by numerical simulations and experiment and paves the way for establishing quantitative methods to determine the atomic structures of small molecules in solution with resolution approaching that of crystallography.
ABSTRACT
Bacterial microcompartments (BMCs) are self-assembling, selectively permeable protein shells that encapsulate enzymes to enhance catalytic efficiency of segments of metabolic pathways through means of confinement. The modular nature of BMC shells' structure and assembly enables programming of shell permeability and underscores their promise in biotechnology engineering efforts for applications in industry, medicine, and clean energy. Realizing this potential requires methods for encapsulation of abiotic molecules, which have been developed here for the first time. We report in vitro cargo loading of BMC shells with ruthenium photosensitizers (RuPS) by two approachesâone involving site-specific covalent labeling and the other driven by diffusion, requiring no specific interactions between cargo molecules and shell proteins. The highly stable shells retain encapsulated cargo over 1 week without egress and preserve RuPS photophysical activity. This study is an important foundation for further work that will converge biological BMC architecture with synthetic chemistry to facilitate biohybrid photocatalysis.
Subject(s)
Photosensitizing Agents , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Ruthenium/chemistryABSTRACT
The application of grazing-incidence total X-ray scattering (GITXS) for pair distribution function (PDF) analysis using >50â keV X-rays from synchrotron light sources has created new opportunities for structural characterization of supported thin films with high resolution. Compared with grazing-incidence wide-angle X-ray scattering, which is only useful for highly ordered materials, GITXS/PDFs expand such analysis to largely disordered or nanostructured materials by examining the atomic pair correlations dependent on the direction relative to the surface of the supporting substrate. A characterization of nanocrystalline In2O3-derived thin films is presented here with in-plane-isotropic and out-of-plane-anisotropic orientational ordering of the atomic structure, each synthesized using different techniques. The atomic orientations of such films are known to vary based on the synthetic conditions. Here, an azimuthal orientational analysis of these films using GITXS with a single incident angle is shown to resolve the markedly different orientations of the atomic structures with respect to the planar support and the different degrees of long-range order, and hence, the terminal surface chemistries. It is anticipated that orientational analysis of GITXS/PDF data will offer opportunities to extend structural analyses of thin films by providing a means to qualitatively determine the major atomic orientation within nanocrystalline and, eventually, non-crystalline films.
ABSTRACT
We have designed two new supramolecular assemblies based on Co(ii)-templated coordination of Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl) analogues as photosensitizers and electron donors to a cobaloxime macrocycle, which are of interest as proton reduction catalysts. The self-assembled photocatalyst precursors were structurally characterized by Co K-edge X-ray absorption spectroscopy and solution-phase X-ray scattering. Visible light excitation of one of the assemblies has yielded instantaneous electron transfer and charge separation to form a transient Co(i) state which persists for 26 ps. The development of a linked photosensitizer-cobaloxime architecture supporting efficient Co(i) charge transfer is significant since it is mechanistically critical as the first photo-induced electron transfer step for hydrogen production, and has not been detected in previous photosensitizer-cobaloxime linked dyad assemblies. X-band EPR spectroscopy has revealed that the Co(ii) centres of both assemblies are high spin, in contrast to most previously described cobaloximes, and likely plays an important role in facilitating photoinduced charge separation. Based on the results obtained from ultrafast and nanosecond transient absorption optical spectroscopies, we propose that charge recombination occurs through multiple ligand states present within the photosensitizer modules. The studies presented here will enhance our understanding of supramolecular photocatalyst assembly and direct new designs for artificial photosynthesis.
ABSTRACT
Developing efficient photocatalysts that perform multi electron redox reactions is critical to achieving solar energy conversion. One can reach this goal by developing systems which mimic natural photosynthesis and exploit strategies such as proton-coupled electron transfer (PCET) to achieve photochemical charge accumulation. We report herein a heteroleptic Cu(i)bis(phenanthroline) complex, Cu-AnQ, featuring a fused phenazine-anthraquinone moiety that photochemically accumulates two electrons in the anthraquinone unit via PCET. Full spectroscopic and electrochemical analyses allowed us to identify the reduced species and revealed that up to three electrons can be accumulated in the phenazine-anthraquinone ring system under electrochemical conditions. Continuous photolysis of Cu-AnQ in the presence of sacrificial electron donor produced doubly reduced monoprotonated photoproduct confirmed unambiguously by X-ray crystallography. Formation of this photoproduct indicates that a PCET process occurred during illumination and two electrons were accumulated in the system. The role of the heteroleptic Cu(i)bis(phenanthroline) moiety participating in the photochemical charge accumulation as a light absorber was evidenced by comparing the photolysis of Cu-AnQ and the free AnQ ligand with less reductive triethylamine as a sacrificial electron donor, in which photogenerated doubly reduced species was observed with Cu-AnQ, but not with the free ligand. The thermodynamic properties of Cu-AnQ were examined by DFT which mapped the probable reaction pathway for photochemical charge accumulation and the capacity for solar energy stored in the process. This study presents a unique system built on earth-abundant transition metal complex to store electrons, and tune the storage of solar energy by the degree of protonation of the electron acceptor.
ABSTRACT
Photodriven charge-transfer dynamics and catalytic properties have been investigated for a hybrid system containing CdSe/ZnS core/shell quantum dots (QDs) and surface-bound molecular cobaloxime catalysts. The electron transfer from light-excited QDs to cobaloxime, revealed by optical transient absorption spectroscopy, takes place with an average time constant of 105 ps, followed a much slower charge recombination process with a time constant of â«3 ns. More interestingly, we also observed photocatalytic hydrogen generation by this QD/cobaloxime hybrid system, with >10,000 turnovers of H(2) per QD in 10 h, using triethanolamine as a sacrificial electron donor. These results suggest that QD/cobaloxime hybrids succeed in coupling single-photon events with multielectron redox catalytic reactions, and such systems could have potential applications in long-lived artificial photosynthetic devices for fuel generation from sunlight.
ABSTRACT
Flavin chemistry is ubiquitous in biological systems with flavoproteins engaged in important redox reactions. In photosynthesis, flavin cofactors are used as electron donors/acceptors to facilitate charge transfer and accumulation for ultimate use in carbon fixation. Following light-induced charge separation in the photosynthetic transmembrane reaction center photosystem I (PSI), an electron is transferred to one of two small soluble shuttle proteins, a ferredoxin (Fd) or a flavodoxin (Fld) (the latter in the condition of Fe-deficiency), followed by electron transfer to the ferredoxin-NADP+ reductase (FNR) enzyme. FNR accepts two of these sequential one electron transfers, with its flavin adenine dinucleotide (FAD) cofactor becoming doubly reduced, forming a hydride which is then passed onto the substrate NADP+ to form NADPH. The two one-electron potentials (oxidized/semiquinone and semiquinone/hydroquinone) are similar to each other with the FNR protein stabilizing the hydroquinone, making spectroscopic detection of the intermediate semiquinone state difficult. We employed a new biohybrid-based strategy that involved truncating the native three-protein electron transfer cascade PSI â Fd â FNR to a two-protein cascade by replacing PSI with a molecular Ru(ii) photosensitizer (RuPS) which is covalently bound to Fd and Fld to form biohybrid complexes that successfully mimic PSI in light-driven NADPH formation. RuFd â FNR and RuFld â FNR electron transfer experiments revealed a notable distinction in photosynthetic charge accumulation that we attribute to the different protein cofactors [2Fe2S] and flavin. After freeze quenching the two-protein systems under illumination, an intermediate semiquinone state of FNR was readily observed with cw X-band EPR spectroscopy. The increased spectral resolution from selective deuteration allowed EPR detection of inter-flavoprotein electron transfer. This work establishes a biohybrid experimental approach for further studies of photosynthetic light-driven electron transfer chain that culminates at FNR and highlights nature's mechanisms that couple single electron transfer chemistry to charge accumulation, providing important insight for the development of photon-to-fuel schemes.