Electron transport chains are crucial for life, involving redox-active metalloproteins with metal cofactors. The first coordination sphere of these cofactors is highly conserved, while surrounding residues modulate their electronic structure, leading to a wide range of reduction potentials that drive electron cascades. The protein matrix also dictates cofactor orientation, regulating electron transfer. Metalloprotein design requires finely tunable redox-active metal sites for photo-induced electron trafficking and bioenergy control. Previous work has focused on charge separation/recombination at abiotic cofactors, electron transfer to natural acceptors, injection into photoanodes, and intra-protein electron transfer. Naturally occurring redox proteins involved in bioenergy control include cupredoxins, cytochromes, and iron-sulfur proteins, with rubredoxins (Rds) being the simplest and most studied. Rds bind a single iron ion through four Cys residues, cycling between oxidation states (II) and (III). Despite conserved backbones and sequences, their reduction potentials vary significantly. This study aims to design a miniaturized rubredoxin with a high reduction potential, to be used as a terminal electron acceptor in an artificial photosynthetic electron transport chain.

Extensive research has explored the design and modification of metalloproteins, particularly focusing on understanding the minimal coordination spheres required for metal binding and activity. Studies on rubredoxins have revealed the importance of hydrogen bonding in the second coordination sphere in influencing the reduction potential of the FeS4 center. Previous attempts at mimicking rubredoxins through de novo design have employed various strategies, including the use of cyclic peptides and dimeric scaffolds. However, this work addresses the challenge of creating a highly tunable redox-active miniprotein through a streamlined design approach, based on the minimized rubredoxin structure. The use of porphyrin-containing proteins as photosensitizers in artificial electron transport chains is also established, but the creation of a fully artificial chain using this approach was the key innovation.

The design strategy involved miniaturizing the *Clostridium pasteurianum* rubredoxin (Cp Rd) V44A mutant structure. A C2 longitudinal axis was applied to the Val38-Glu50 fragment to generate dimer coordinates. A systematic search identified a four-residue loop to link the N- and C-termini, favoring Gly residues in β-turn motifs. The best-matching fragment was grafted onto the dimer, and further backbone design refined the structure, incorporating Aib residues to induce 310-helix formation. A second design round optimized side-chain packing, and selected hydrophilic residues for the CXXC motif. The resulting 28-residue miniprotein, METPsc1, was synthesized by solid-phase peptide synthesis. The zinc complex (ZnMETPsc1) was characterized by X-ray diffraction, revealing high similarity to the designed model (backbone RMSD 0.45 Å). Spectroscopic techniques, including UV-Vis, CD, and EPR, characterized the iron complex (FeMETPsc1), confirming tetrahedral iron coordination by four Cys residues and high-spin Fe3+ center. Redox cycling experiments demonstrated reversible switching between ferrous and ferric states. Cyclic voltammetry determined the reduction potential (E0 = 121 mV vs SHE), significantly higher than prokaryotic Rds. Finally, a photo-triggered reduction experiment using ZnMC6*a as a photosensitizer and TEA as a sacrificial reductant demonstrated electron transfer from the photosensitizer to FeMETPsc1 upon green light irradiation.

The study successfully designed and synthesized a 28-residue miniprotein, METPsc1, with high structural fidelity to the designed model (0.45 Å backbone RMSD). The zinc complex of METPsc1 exhibits a tetrahedral coordination geometry, consistent with natural rubredoxins. The iron complex (FeMETPsc1) showed reversible redox cycling between ferrous and ferric states, indicating functional similarity to native rubredoxins. Importantly, FeMETPsc1 demonstrated a high reduction potential (121 mV vs SHE), exceeding the typical range for prokaryotic rubredoxins. The high reduction potential of FeMETPsc1 enabled it to function as the terminal electron acceptor in a fully artificial electron transport chain, triggered by visible light. This chain utilized ZnMC6*a as a photosensitizer and TEA as a sacrificial electron donor. Upon green light irradiation, reduction of Fe3+METPsc1 was observed, confirming the successful operation of the artificial electron chain. However, repeated cycles led to partial reduction and ZnMC6*a degradation, possibly due to reactive oxygen species or radical formation.

The successful design and characterization of METPsc1 validate the design strategy and demonstrate the feasibility of creating miniature redox proteins with predetermined properties. The high reduction potential of FeMETPsc1 is attributed to specific second-shell interactions, highlighting the importance of precise structural design in achieving desired redox properties. The creation of a fully artificial photosynthetic electron transport chain demonstrates the potential of using de novo-designed miniproteins to build functional biological devices. While the photo-induced electron transfer showed initial success, the observed degradation of ZnMC6*a during repeated cycles highlights the need for further optimization to enhance stability and efficiency.

This work presents a generalizable methodology for designing miniature redox proteins, exemplified by the creation of a high-potential rubredoxin mimic, METPsc1. The sub-Å agreement between the designed and experimental structures validates the design principles. The successful integration of FeMETPsc1 into a fully artificial photo-triggered electron transport chain opens avenues for constructing sophisticated nanosized multicomponent mini-protein devices. Future research could focus on enhancing the stability of the photosensitizer and optimizing the electron transport chain for greater efficiency and long-term stability.

The artificial electron transport chain demonstrated only a few successful reduction cycles before the photosensitizer (ZnMC6*a) degraded. This limitation necessitates further investigation into the stability of the photosensitizer and the potential for reactive oxygen species formation within the system. The study focused on a single photosensitizer and electron donor; exploring alternative components may improve performance. The overall miniaturization strategy, while successful, might not be universally applicable to other redox proteins requiring different structural elements for their function.