Here, we report the development of a novel photoactive biomolecular nanoarchitecture based on the genetically engineered extremophilic photosystem I (PSI) biophotocatalyst interfaced with a single layer graphene via pyrene-nitrilotriacetic acid self-assembled monolayer (SAM). For the oriented and stable immobilization of the PSI biophotocatalyst, an His₆-tag was genetically engineered at the N-terminus of the stromal PsaD subunit of PSI, allowing for the preferential binding of this photoactive complex with its reducing side towards the graphene monolayer. This approach yielded a novel robust and ordered nanoarchitecture designed to generate an efficient direct electron transfer pathway between graphene, the metal redox center in the organic SAM and the photo-oxidized PSI biocatalyst. The nanosystem yielded an overall current output of 16.5 µA·cm⁻² for the nickel- and 17.3 µA·cm⁻² for the cobalt-based nanoassemblies, and was stable for at least 1 h of continuous standard illumination. The novel green nanosystem described in this work carries the high potential for future applications due to its robustness, highly ordered and simple architecture characterized by the high biophotocatalyst loading as well as simplicity of manufacturing.

The construction of an efficient conductive interface between electrodes and electroactive proteins is a major challenge in the biosensor and bioelectrochemistry fields to achieve the desired nanodevice performance. Concomitantly, metallo-organic terpyridine wires have been extensively studied for their great ability to mediate electron transfer over a long-range distance. In this study, we report a novel stepwise bottom-up approach for assembling bioelectrodes based on a genetically modified model electroactive protein, cytochrome c₅₅₃ (cyt c₅₅₃) and an organometallic terpyridine (TPY) molecular wire self-assembled monolayer (SAM). Efficient anchoring of the TPY derivative (TPY-PO(OH)₂) onto the ITO surface was achieved by optimising solvent composition. Uniform surface coverage with the electroactive protein was achieved by binding the cyt c₅₅₃ molecules via the C-terminal His₆-tag to the modified TPY macromolecules containing Earth abundant metallic redox centres. Photoelectrochemical characterisation demonstrates the crucial importance of the metal redox centre for the determination of the desired electron transfer properties between cyt and the ITO electrode. Even without the cyt protein, the ITO-TPY nanosystem reported here generates photocurrents whose densities are 2-fold higher that those reported earlier for ITO electrodes functionalised with the photoactive proteins such as photosystem I in the presence of an external mediator, and 30-fold higher than that of the pristine ITO. The universal chemical platform for anchoring and nanostructuring of (photo)electroactive proteins reported in this study provides a major advancement for the construction of efficient (bio)molecular systems requiring a high degree of precise supramolecular organisation as well as efficient charge transfer between (photo)redox-active molecular components and various types of electrode materials.

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec553(cytc553)peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc553holoprotein and thesurface anchoring C-terminal His6-tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers.

Photosystem I (PSI)‐based photovoltaic performance is hampered by short‐circuiting and charge recombination between randomly oriented PSI complexes on the electrode surface. Various types of molecular interfaces have been designed to introduce control over the order of the PSI biophotocatalyst in the biomolecular nanodevices; yet, the precise orientation of the PSI complex has never been directly shown and quantified. Here, cryo‐electron microscopy (cryo‐EM) visualization of PSI particles following domain‐specific anchoring of this complex to the single‐layer graphene (SLG) is reported. By applying the rational design of the biomolecular interface, the preferential orientation of the PSI complex with its reducing side toward the SLG is directly demonstrated. This approach is effective to enhance anodic photocurrent generation three‐fold compared to the nanosystem with randomly deposited PSI particles. The cryo‐EM visualization of the PSI biomolecules on the graphene electrode surface provides the validation of the rational design of photocatalytic nanoarchitecture aimed at significantly improved photochemical performance.

Construction of green nanodevices characterised by excellent long-term performance remains high priority in biotechnology and medicine. Tight electronic coupling of proteins to electrodes is essential for efficient direct electron transfer (DET) across the bio-organic interface. Rational modulation of this coupling depends on in-depth understanding of the intricate properties of interfacial DET. Here, we dissect the molecular mechanism of DET in a hybrid nanodevice in which a model electroactive protein, cytochrome c₅₅₃ (cyt c₅₅₃), naturally interacting with photosystem I, was interfaced with single layer graphene (SLG) via the conductive self-assembled monolayer (SAM) formed by pyrene–nitrilotriacetic acid (pyr–NTA) molecules chelated to transition metal redox centers. We demonstrate that efficient DET occurs between graphene and cyt c₅₅₃ whose kinetics and directionality depends on the metal incorporated into the bio-organic interface: Co enhances the cathodic current from SLG to haem, whereas Ni exerts the opposite effect. QM/MM simulations yield the mechanistic model of interfacial DET based on either tunnelling or hopping of electrons between graphene, pyr–NTA–M²⁺ SAM and cyt c₅₅₃ depending on the metal in SAM. Considerably different electronic configurations were identified for the interfacial metal redox centers: a closed-shell system for Ni and a radical system for the Co with altered occupancy of HOMO/LUMO levels. The feasibility of fine-tuning the electronic properties of the bio-molecular SAM upon incorporation of various metal centers paves the way for the rational design of the optimal molecular interface between abiotic and biotic components of the viable green hybrid devices, e.g. solar cells, optoelectronic nanosystems and solar-to-fuel assemblies.

Solar-converting nanosystems using self-renewing biomaterial resources carry great potential for developing sustainable technologies to ameliorate climate change and minimize reliance on fossil fuels. By mimicking natural photosynthesis, diverse proof-of-concept biosolar systems have been used to produce green electricity, fuels and chemicals. Efforts so far have focused on optimizing light harvesting, biocatalyst loading and electron transfer (ET), however, the long-term performance of best-performing systems remains a major challenge due to the intensive use of diffusive, toxic mediators. To overcome this limitation, we developed a rationally designed nanosystem based on the entrapment of non-toxic mediator, ferrocene dimethanol (Fc), localized at the abiotic–biotic molecular interface that efficiently promoted ET between electrode surface and two photosynthetic proteins: cytochrome c and photosystem I. We demonstrate that space-confined Fc mediators (1 nM) are as effective in terms of ET kinetics as a 500 000-fold higher concentration of freely-diffusive Fc. The Fc-confined biophotocathodes showed a milestone photocurrent density of 14 μA cm⁻² under oxic conditions compared to analogous planar (2D) biophotoelectrodes, with a photoconductive biolayer stable for over 5 months. The space-confined ET mediation reported in this work opens a new avenue for efficiently interfacing biomachineries, providing a benchmark design advancement in the quest for viable biohybrid technologies.