Photodynamics of Photo-Activated BLUF Coupled Endonuclease III Mutant RmPAE from Mesophilic, Pigmented Bacterium Rubellimicrobium mesophilum Strain MSL-20T

The pink to light reddish-pigmented bacterium Rubellimicrobium mesophilum strain MSL-20T contains a BLUF coupled endonuclease III of unknown function. A purified recombinant triple mutated sample of the BLUF coupled endonuclease III (F5Y, N27H, A87W) named RmPAE (Rubellimicrobium mesophilum PhotoActivated Endonuclease) was produced and characterized by optical spectroscopic methods. The BLUF domain photo-cycling dynamics occurred with high efficient blue-light induced signaling state formation (quantum yield of signaling state formation φs ≈ 0.6), small spectral red-shift (δλs,r ≈ 5.4 nm), and slow thermal activated dark recovery to the receptor state (τrec ≈ 20 min at room temperature). An apparent RmPAE melting temperature of θm ≈ 63 °C was determined by stepwise sample heating and absorption spectrum analysis. The photo-degradation of RmPAE in the signaling state was determined by prolonged intense bluelight exposure. An irreversible flavin photo-degradation occurred with quantum yield of φD ≈ 2.6×10 -5. Schemes of the photo-cycling and the photo-degradation dynamics are presented. Engineered RmPAE may find application as light guided DNA cutter in optogenetic applications.