Photo-dynamics of the lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain

•Lyophilizing of NgPAC2 from Naegleria gruberi caused loss of BLUF domain activity.•Photo-induced tyrosine to flavin electron transfer in lyophilized NgPAC2.•Photo-induced Tyr-Tyr cross-linking to o,o′-dityrosine in lyophilized NgPAC2.•Photo-induced partial flavin cofactor reduction in lyophilized NgPAC2.•Two NgPAC2 conformations with fast and slow photo-induced electron transfer.

The absorption and emission spectroscopic behavior of lyophilized photo-activated adenylate cyclase NgPAC2 from the amoeboflagellate Naegleria gruberi NEG-M strain consisting of a BLUF domain (BLUF = Blue Light sensor Using Flavin) and a cyclase homology domain was studied in the dark, during blue-light exposure and after blue-light exposure at a temperature of 4 °C. The BLUF domain photo-cycle dynamics observed for snap-frozen NgPAC2 was lost by lyophilization (no signaling state formation with flavin absorption red-shift). Instead, blue-light photo-excitation of lyophilized NgPAC2 caused sterically restricted Tyr-Tyr cross-linking (o,o′-ditysosine formation) and partial flavin cofactor reduction.

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