Abstract
Abstract The absorption and emission spectroscopic behavior of the photo-activated adenylyl cyclase NgPAC3 from the amoeboflagellate Naegleria gruberi NEG-M strain was studied. The flavin cofactor was found to be partly fully oxidized and partly fully reduced. The typical BLUF domain (BLUF = B lue L ight sensor U sing F lavin) oxidized flavin absorption photo-cycle dynamics with about 14 nm flavin absorption red-shift in the signaling state was observed. The quantum efficiency of signaling state formation was determined to be ϕ s = 0.66 ± 0.03. A bi-exponential signaling state recovery to the dark-adapted receptor state was observed with the time constants τ rec,f = 275 s and τ rec,sl = 45 min. The thermal irreversible protein unfolding was studied and an apparent protein melting temperature of ϑ m ≈ 50 °C was found. The photodynamic behavior of NgPAC3 is compared with the behavior of the previously investigated photo-activated cyclases NgPAC1 (nPAC) and NgPAC2 from the same N. gruberi NEG-M strain. Purified recombinant NgPAC3 showed light-gated adenylate cyclase activity upon illumination with blue light. Its cyclase activity is compared with those of NgPAC1 and NgPAC2.
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