Cryo-electron microscopy confirms that controlled alignment of Photosystem I on graphene boosts electron flow and durability in biohybrid photoanodes, linking molecular order to improved solar energy conversion.
(Nanowerk Spotlight) Artificial photosynthesis aims to replicate the process by which plants capture sunlight and convert it into chemical energy. In nature, this transformation is managed with exquisite precision inside chloroplasts, where specialized protein complexes handle the movement of electrons with almost no loss.
Translating that biological efficiency into artificial devices has remained difficult. The main obstacle is not in harvesting light but in controlling how the biological components connect to man-made materials.
Photosystem I, or PSI, a key protein in natural photosynthesis, can generate long-lived charge separation when illuminated. That property makes it a promising candidate for hybrid solar devices, but its performance outside the cell depends on orientation. When PSI attaches to a conductive surface in a random or sideways position, electrons may take short paths that cause energy to dissipate.
Researchers have relied on indirect indicators such as photocurrent or surface potential to infer how PSI sits on an electrode. The lack of direct visualization has prevented precise design.
This limitation has begun to change thanks to advances in cryogenic electron microscopy, or cryo-EM, which can image biological structures in their native hydrated state at near-atomic resolution.
At the same time, single-layer graphene has matured as a transparent, conductive, and chemically stable support suitable for both imaging and photoelectrochemical applications. These tools now allow scientists to see, rather than guess, how proteins organize on conductive substrates.
The researchers combined genetic modification of PSI with targeted surface chemistry to favor one specific alignment, then verified the outcome through direct imaging. Their goal was to tether the electron-emitting side of PSI to graphene while keeping the electron-receiving side exposed to the solution, mimicking the geometry of natural photosynthesis.
Electron transfer pathways in biomolecular nanosystems. A) Bioelectrode with randomly oriented PSI molecules (one of the possible orientations shown). B) PSI-based biophotoanode. C) PSI-based biophotocathode. Depicted are: the main electron transfer (gray arrows) and short-circuiting and charge recombination electron pathways (blue arrows). Molecular wiring of His-tagged PSI biophotocatalyst with the single-layer graphene (SLG) surface via metalorganic self-asembled monolayer (SAM) of pyrene- nitrilotriacetic acid (NTA)-M2+ molecules, is shown. (Image: Reproduced from DOI:10.1002/adfm.202510926, CC BY)
PSI is a large protein complex that absorbs light through chlorophyll molecules near its reaction center, called P700. When excited by light, P700 becomes oxidized, while a small iron–sulfur cluster named FB at the opposite end becomes reduced. In living cells, electrons flow from FB through soluble carriers to generate chemical fuels, while P700 is re-supplied with electrons from other parts of the photosynthetic system. When the protein is placed on an electrode, orientation determines whether electrons are extracted efficiently or lost through recombination.
To control orientation, the team introduced a short chain of six histidine amino acids, known as a His6 tag, onto the PsaD subunit located near the FB site. Histidines form stable bonds with nickel ions.
Using this property, the researchers coated single-layer graphene with a self-assembled layer of pyrene nitrilotriacetic acid (pyrene-NTA) containing nickel. The pyrene ring adheres to graphene through strong aromatic interactions, while the nitrilotriacetic acid coordinates nickel, which in turn binds the histidine tag.
This configuration creates a molecular bridge that should pull PSI into a specific orientation, positioning its reducing side toward the surface. A control sample used identical graphene and surface treatment but with untagged PSI, leading to random attachment driven by nonspecific interactions.
Cryo-EM imaging confirmed that these design choices influenced orientation. Hundreds of micrographs were collected and analyzed using single-particle reconstruction methods. On untreated graphene, native PSI was found to lie flat, with its side pressed against the surface. This configuration places both the oxidized and reduced ends of the complex at similar distances from the electrode, a geometry expected to favor short-circuiting and inefficient charge transfer.
On nickel-functionalized graphene, His6-tagged PSI showed a different arrangement. Roughly 40 percent of the complexes were oriented with their electron-emitting side facing the surface, consistent with the intended binding through the nickel–histidine interaction. The rest still lay laterally, suggesting that nonspecific adsorption remained competitive.
A second data set produced nearly identical statistics, confirming reproducibility. These measurements represent the first direct, quantitative observation of partial alignment achieved by rational molecular design rather than by chance.
Having established the structure, the researchers examined its functional consequences. They fabricated photoelectrodes using fluorine-doped tin oxide coated with single-layer graphene treated with the same chemistry as the cryo-EM samples.
The devices operated as photoanodes, meaning electrons were drawn from the FB side of PSI into the electrode. To close the circuit, sodium ascorbate served as an external electron donor, regenerating oxidized P700. Under light at an applied potential of +1.11 volts versus the reversible hydrogen electrode, the oriented PSI electrodes produced substantially higher anodic photocurrent than the randomly adsorbed controls.
After one hour of continuous illumination, the native PSI electrode yielded 49.6 microamperes per square centimeter, while the oriented version reached 150 microamperes per square centimeter. The threefold increase directly linked orientation to functional output.
Further tests identified the specific electron pathways involved. When oxygen was removed, photocathodic currents at negative potentials decreased, confirming its role as an electron acceptor under those conditions. When sodium ascorbate was omitted at positive bias, the anodic current dropped from 8.43 to 0.9 microamperes per square centimeter, showing that replenishing electrons to P700 is necessary for sustained operation.
These experiments demonstrate that the designed interface promotes a clear, one-way electron flow from the reducing side of PSI into the graphene electrode.
Durability proved equally important. The oriented electrodes maintained 147 microamperes per square centimeter for four hours of continuous illumination at +1.11 volts in the presence of sodium ascorbate. After sixteen days of dark storage at 4 °C, the same electrodes lost only sixteen percent of their activity during a one-hour test.
This level of stability indicates that both the protein and the nickel-chelate linker remain intact through repeated cycles of light exposure, addressing one of the main weaknesses of earlier PSI devices.
The study’s main advance is methodological as much as practical. It demonstrates that cryo-EM can reveal the molecular organization of biohybrid materials directly on conductive substrates, turning structural imaging into a design tool for energy devices.
The confirmed link between protein alignment and photocurrent provides a clear engineering rule: position the electron-emitting face of PSI toward the electrode using a metal-binding tag and a compatible chelating linker. This approach can be generalized to other redox enzymes and catalytic proteins that require specific orientation for efficient charge transfer.
Artificial photosynthesis remains a developing field, but this work resolves a major uncertainty. By showing that structural order at the molecular scale translates into measurable electronic performance, it bridges biological precision and material engineering.
With tools such as cryo-EM and graphene-based interfaces, researchers can refine surface chemistry, adjust tag placement, and explore new coupling strategies that guide electrons with minimal loss. Direct visualization now replaces assumption, providing a measurable path toward efficient and stable solar-to-chemical conversion systems.
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