Design of electron transport chains coupling photosystem I to reductases for light-driven hydrogen production and CO2 reduction-

• Applicant: Martin Winkler, Ph.D.
• Subject Area: Biochemistry ; Biophysics ; Structural Biology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 561053011

This research project aims to identify and resolve molecular bottlenecks in the coupling of Photosystem I (PSI) with redox enzymes in biophotovoltaic systems. To this end, we will study a PSI-based model photocathode with [FeFe]-hydrogenase and FDH as catalysts for hydrogen production and CO₂ reduction. We will leverage the native ETCs of these reductases by coupling them to PSI's reducing end. This will be achieved by using state-of-the-art deep-learning protein design algorithms to redesign the reductases to have precise binding interfaces to the stromal ridge of PSI, which natively serves as the binding site of PSI's terminal electron acceptor. Specifically, the design targets will be bacterioferredoxin domains that are part of the [FeFe]-hydrogenase DdHydAB from Desulfovibrio desulfuricans, and the HycB subunit that is directly coupled to ClFdhF, the catalytic domain of FDH from Clostridium ljungdahlii. The redesigned interfaces will be evaluated for binding, self-assembly, electron transfer efficiency, and catalytic performance in solution and in biophotovoltaic systems. As a reference for photohydrogen evolution, PSI's natural hydrogen production catalyst, the [FeFe]-hydrogenase CrHydA1 of the unicellular green alga Chlamydomonas reinhardtii will be assessed. Our teams will iteratively refine designs of the reductases' binding interfaces to PSI. Using in silico models, we will implement targeted mutagenesis around these interfaces to compare wild-type and redesigned enzyme variants in terms of iron-sulfur cluster content, enzyme kinetics, and retention of enzymatic features. These variants will be tested for PSI coupling efficiency to improve photo-driven H2 and CO2 reduction. The assessment of the final assembly will comprise photocatalytic activity (turnover frequency, turnover number, and quantum yields) in solution and in immobilized configurations, with PSI-coupled variants embedded in a conductive redox hydrogel matrix.

• DFG Programme: Research Grants
• International Connection: Israel
• Partner Organisation: The Israel Science Foundation
• Cooperation Partner: Professor Dror Noy, Ph.D.