Photoreaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes.

The photochemical reaction of photoactivated adenylate cyclase from cyanobacterium Microcoleus chthonoplastes PCC 7420 (mPAC), which consists of a Per-Arnt-Sim (PAS), a light‑oxygene-voltage (LOV), and an adenylate cyclase (AC) domain, was investigated mainly using the time-resolved transient grating method. An absorption spectral change associated with an adduct formation between its chromophore (flavin mononucleotide) and a cysteine residue was observed with a time constant of 0.66 μs. After this reaction, a significant diffusion coefficient (D)-change was observed with a time constant of 38 ms. The determined D-value was concentration-dependent indicating a rapid equilibrium between the dimer and tetramer. Combining the results of size exclusion chromatography and CD spectroscopy, we concluded that the photoinduced D-change was mainly attributed to the equilibrium shift from the dimer rich to the tetramer rich states upon light exposure. Since the reaction rate does not depend on concentration, the rate determining step of the tetramer formation is not the collision of proteins by diffusion, but a conformation change. The roles of the PAS and AC domains as well as the N- and C-terminal flanking helices of the LOV domain (A'α- and Jα-helices) were investigated using various truncated mutants. The PAS domain was found to be a strong dimerization site and is related to efficient signal transduction. It was found that simultaneous existence of the A'α- and Jα-helices in mPAC is important for the light-induced conformation change to lead the conformation change which induces the tetramer formation. The results suggest that the angle changes of the coiled-coil structures in the A'α and Jα-helices are essential for this conformation change. The reaction scheme of mPAC is proposed.