BPS2026 – Evolutionary plasticity in BLUF domain creates first natural light-gated endonuclease with enhanced catalytic activity

The BLUF-endonuclease (BLUF-endo) from Rubellimicrobium mesophilum contains naturally occurring mutations (Y5F/H27N/W87A) at positions critical for canonical BLUF photochemistry. We investigated how these evolutionary changes affect photodynamics and light-gated endonuclease function, revealing an unprecedented photoactivation mechanism. Spectroscopic characterization of BLUF-endo triple mutant (TM) wild-type demonstrated complete disruption of classical BLUF photophysics; absence of the characteristic 10 nm red-shift, altered dark-state recovery (τ = 687 s vs. 462 s for wild-type), and 15-fold increased fluorescence lifetime (2.02 ns vs. 0.13 ns). Circular dichroism confirmed loss of light-induced conformational changes in TM variant. Molecular dynamics simulations (100 ns) revealed decreased structural stability (RMSD: 0.152 nm vs. 0.124 nm) and unexpected domain flipping upon FAD binding, suggesting alternative photosignaling pathways of BLUF domain. Remarkably, despite unconventional photodynamics, the BLUF-endo (TM) exhibited enhanced light-induced endonuclease activity. The TM fluorescence quenching studies confirmed rapid DNA binding (Kq = 3.4×10 12 M -1 s -1 ). Our findings establish that evolutionary divergence from canonical BLUF architecture creates an alternative electron-transfer pathway, enhancing endonuclease enzymatic efficiency while maintaining photoregulation. This represents the first natural light-gated endonuclease—potentially functioning as an optically controlled bacterial defence mechanism against phage invasion via cleavage of viral DNA. This discovery opens new avenues for engineering optogenetic tools and understanding photoreceptor evolution in bacterial immunity systems.