床次 俊郎

Abstract The Orange Carotenoid Protein (OCP) is a blue-green light sensor that regulates non-photochemical quenching (NPQ) in cyanobacteria through reversible transitions between its dark-adapted (OCP^O) and light-adapted (OCP^R) states. Despite extensive studies, the detailed reaction scheme remains unclear. In this study, we examined the photo-induced reaction dynamics of OCP using size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and transient grating (TG) spectroscopy. We found that OCP^Oand OCP^Rexist in monomer–dimer equilibria, with OCP^Rforming more stable and elongated dimers. TG measurements revealed that upon photoexcitation, OCP^Omonomers undergo two structural transitions before associating into OCP^Rdimers. In contrast, OCP^Odimers dissociate prior to the structural rearrangement, highlighting a fundamental difference in their reaction pathways. Moreover, dimerization was found to moderately reduce the photo-reactivity of OCP^Ocompared to the monomer. We also found that apo-OCP readily forms heterodimers with OCP^R, potentially altering reaction pathways and masking true kinetic behavior.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC (FL-PAC) and the BLUF domain with the α3-helix. DSCC of FL-PAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.