Electrocatalytic- and solar-driven fuel synthesis from the greenhouse gas CO2 is a desirable approach to simultaneously produce sustainable energy carriers, and combat increasing atmospheric CO2 levels. Formate is a stable intermediate in the reduction of CO2 and is utilised in a wide range of downstream applications. Recent efforts have focused on using an all-protein, light-triggered, catalytic circuit based on photosystem I (PSI), cytochrome c6 (cyt c₆ ) and formate dehydrogenase (FDH), which would convert CO2 into formate. However, various challenges remained. Our research addresses the optimization of the structural basis for efficient electron transfer from cyt c₆ to a genetically engineered PSI-FDH fusion complex. Due to the transient binding of cyt c₆ to PSI it has been challenging to investigate the structural basis for this binding process and optimise binding affinities (1,2). Structural observations and models propose a specific binding site for cyt c₆ near the P700 chlorophyll pair of PSI on the lumenal side (1). Based on this model, residues likely involved in the binding mechanism were predicted. Here, heterologously expressed cyt c₆ variants from the cyanobacterium Thermosynechococcus vestitus BP-1 (previously known as T. elongatus) with a higher binding affinity to this suggested binding site were used to study the binding mechanism between cyt c₆ and PSI. These high affinity variants show increased oxygen reduction rates compared to the wildtype. Based on these results, the binding mechanism will be further specified via Isothermal Titration Calorimetry (ITC) and structural determination of the cyt c₆ -PSI complex by cryo-electron microscopy (cryo-EM).

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