No abstract

Photosystem I (PSI) is a very large membrane protein complex (∼1000 kDa) harboring P700*, the strongest reductant known in biological systems, which is responsible for driving NAD(P) + and ultimately for CO 2 reduction. Although PSI is one of the most important components in the photosynthesis machinery, it has remained difficult to enhance PSI functions through genetic engineering due to its enormous complexity. Inspired by PSI's ability to undergo multiple-step photo-induced electron hopping from P700* to iron−sulfur [Fe 4 S 4 ] clusters, we designed a 33 kDa miniature photocatalytic CO 2 -reducing enzyme (mPCE) harboring a chromophore (BpC) and two [Fe 4 S 4 ] clusters (Fe A /Fe B ). Through reduction potential fine-tuning, we optimized the multiple-step electron hopping from BpC to Fe A /Fe B , culminating in a CO 2 / HCOOH conversion quantum efficiency of 1.43%. As mPCE can be overexpressed with a high yield in Escherichia coli cells without requiring synthetic cofactors, further development along this route may result in rapid photo-enzyme quantum yield improvement and functional expansion through an efficient directed evolution process. KEYWORDS: photosystem I, rational design, photosensitizer protein, iron−sulfur (Fe 4 S 4 ) cluster, photocatalytic, CO 2 reductase