Characterization of the Blue-Light-Activated Adenylyl Cyclase mPAC by Flash Photolysis and FTIR Spectroscopy.

Abstract

The recently discovered photo-activated adenylyl cyclase (mPAC from Microcoleus chthonoplastes) is the first PAC that owes a light-, oxygen- and voltage-sensitive (LOV) domain for blue-light sensing. The photoreaction of the mPAC receptor was studied by time-resolved UV/vis and light-induced Fourier transform infrared (FTIR) absorption difference spectroscopy. The photocycle comprises of the typical triplet state LOV715 and the thio-adduct state LOV390 . While the adduct state decays with a time constant of 8s, the lifetime of the triplet state is with 656ns significantly shorter than in all other reported LOV domains. The light-induced FTIR difference spectrum shows the typical bands of the LOV390 and LOV450 intermediates. The negative S-H stretching vibration at 2573cm-1 is asymmetric suggesting two rotamer configurations of the protonated side chain of C194. A positive band at 3632cm-1 is observed, which is assigned to an internal water molecule. In contrast to other LOV domains, mPAC exhibits a second positive feature at 3674cm-1 which is due to the O-H stretch of a second intrinsic water molecule and the side chain of Y476. We conclude that the latter might be involved in the dimerization of the cyclase domain which is crucial for ATP binding.