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 photodynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Bluelight exposure of nPAC caused a typical BLUF-type photo-cycle behavior (spectral absorption ...
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 photodynamics of the nPAC protein was studied by optical absorption and fluorescence spectroscopy. Bluelight 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(center dot)). A photo-induced electron transfer from Tyr or Trp to flavin (Tyr(+)-Fl(center dot) or Trp(center dot+)- Fl(center dot)radical anion-Fl radical anion or Tyr radical anion-Fl radical anion 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 Flox to lumichrome was observed. A model of the photo-dynamics of nPAC is developed. (C) 2011 Elsevier B.V. All rights reserved.
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