Design and characterization of porphyrin-based photosensitizing metalloproteins integrated with artificial metalloenzymes for photocatalytic hydrogen production

Hydrogen is regarded as a promising alternative to fossil fuels. A desirable method of its generation is via photocatalysis, combining photosensitizers and hydrogen-evolution catalysts in the presence of an electron donor. Inspired by natural photosynthesis, we designed photosensitizing artificial metalloproteins (ArMs) and integrated them with ArM-based catalysts for photocatalytic hydrogen production from water. Metal porphyrins based on protoporphyrin IX (PPIX) were employed as they are naturally abundant and are effective both as photosensitizers and hydrogen-evolution catalysts. Photosensitizing proteins were created by binding zinc (Zn)PPIX or ruthenium (Ru)PPIX to the haem acquisition system A from Pseudomonas aeruginosa (HasAp). The photosensitizer ArMs were combined with cobalt (Co)PPIX-myoglobin (Mb) or free CoPPIX as hydrogen evolution catalysts. We found that free CoPPIX could replace ZnPPIX or RuPPIX in HasAp, forming CoPPIX-HasAp or RuPPIX-CoPPIX-HasAp complexes with enhanced stability compared to CoPPIX-Mb. Photocatalytic hydrogen production was achieved upon irradiation at 435 nm (ZnPPIX) or 385 nm (RuPPIX), using methyl viologen as an electron carrier and triethanolamine as an electron donor. The ZnPPIX-HasAp/CoPPIX-HasAp system remained intact and active for approximately 42 h, while Ru-based systems that were excited by UV light, exhibited signs of protein cleavage upon prolonged irradiation. These results demonstrate the potential of integrating porphyrin-based ArMs for photosensitization and hydrogen evolution, with HasAp providing a robust scaffold for sustained photocatalytic activity.