Photo-dynamics of photoactivated adenylyl cyclase LiPAC

The photoactivated adenylyl cyclase from the spirochete bacterium Leptonema illini, abbreviated LiPAC, was synthesized and characterized by absorption and fluorescence spectroscopic methods. LiPAC consists of a BLUF (Blue Light sensor Using Flavin) domain and an adenylyl cyclase homology domain (CHD). Photo-excitation of fully oxidized flavin Flox in LiPAC resulted in a typical primary (dark-adapted) BLUF domain photo-cycle dynamics. The quantum efficiency of BLUF domain signaling state formation was determined to be φs ≈ 0.60. Continued blue-lightexcitation of LiPAC in the light-adapted state caused irreversible photo-degradation of noncovalently bound Flox to covalently bound fully reduced flavin Flred with a quantum efficiency of φD ≈ 1.1 × 10. At 20 °C the time constant of signaling state recovery to the receptor state after excitation light switch-off was τrec ≈ 2.6 s. The protein thermal stability was studied by stepwise sample heating and cooling. An apparent LiPAC melting temperature of 54 °C was determined. Schemes of the primary BLUF domain photocycling dynamics and the secondary BLUF domain photo-degradation in the signaling state are presented.