Differentiation of photocycle characteristics of flavin-binding BLUF domains of alpha- and beta-subunits of photoactivated adenylyl cyclase of Euglena gracilis

Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PAC alpha and PAC beta), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PAC alpha F2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PAC beta F2 domain, which like PAC alpha F2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PAC beta F2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PAC alpha F2 were 0.28 0.32 and 34 44 s. The remarkable differences between PAC alpha F2 and PAC beta F2 may be related to the sensitivity of the photoactivation. In PAC alpha F2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PAC beta F2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PAC beta F2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PAC beta F2 plays a main role in suppressing the quantum efficiency.