Abstract
The amoeboflagellate Naegleria gruberi NEG-M comprises a BLUF ( blue light sensor using flavin) regulated adenylate cyclase (nPAC). The nPAC gene was expressed heterologously in Escherichia coli and the photo-dynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Blue-light exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption red-shift, fluorescence quenching, absorption and fluorescence recovery in the dark). Additionally, time-delayed reversible photo-induced one-electron reduction of fully oxidized flavin (Fl ox) to semi-reduced flavin (FlH ) occurred. Furthermore, photo-excitation of FlH caused irreversible electron transfer to fully reduced anionic flavin (FlH −). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr + –Fl − or Trp + –Fl − radical ion-pair formation) is thought to cause H-bond restructuring responsible for BLUF-type photo-cycling and permanent protein re-conformation enabling photo-induced flavin reduction by proton transfer. Some photo-degradation of Fl ox to lumichrome was observed. A model of the photo-dynamics of nPAC is developed.
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