Unfolding of the C‑Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a β-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D₂O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 μs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 μs, along with hydrogen-bond rupture of the FMN C4O with Gln-513, motion of the β-sheet, and an additional helical element. The second phase occurs in approximately 240 μs. The final spectrum at 500 μs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale

Role of PufX in Photochemical Charge Separation in the RC-LH1 Complex from Rhodobacter sphaeroides: An Ultrafast Mid-IR Pump–Probe Investigation

Photochemical charge separation in isolated reaction center-light harvesting 1 (RC-LH1) complexes from Rhodobacter sphaeroides was examined using time-resolved mid-infrared pump–probe spectroscopy. Absorption difference spectra were recorded between 1760 and 1610 cm^–1 with subpicosecond time resolution to characterize excited-state and radical pair dynamics in these complexes, via the induced absorption changes in the keto carbonyl modes of the bacteriochlorophylls and bacteriopheophytins. Experiments on RC-LH1 complexes with and without the polypeptide PufX show that its presence is required to achieve generation of the radical pair P⁺Q_A^– under mildly reducing conditions. In the presence of PufX, the final radical pair formed over a ∼3 ns period was P⁺Q_A^–, but in its absence the corresponding radical pair was P⁺H_A^–, implying that Q_A was either absent in these PufX-deficient complexes or was prereduced. However, P⁺Q_A^– could be generated in PufX-deficient complexes following addition of the oxidant DMSO, showing that Q_A was present in these complexes and allowing the conclusion that under mildly reducing conditions charge separation was blocked after P⁺H_A^– due to the presence of an electron on Q_A. The data provide strong support for the hypothesis that one of the functions of PufX is to regulate the stability of Q_B^–, ensuring the oxidation of Q_A^– in the presence of a reduced quinone pool and so preserving efficient photochemical charge separation under anaerobic conditions

Photoionization and Electron Radical Recombination Dynamics in Photoactive Yellow Protein Investigated by Ultrafast Spectroscopy in the Visible and Near-Infrared Spectral Region

Photoinduced ionization of the chromophore inside photoactive yellow protein (PYP) was investigated by ultrafast spectroscopy in the visible and near-infrared spectral regions. An absorption band that extended from around 550 to 850 nm was observed and ascribed to solvated electrons, ejected from the <i>p</i>-hydroxycinnamic acid anion chromophore upon the absorption of two 400 nm photons. Global kinetic analysis showed that the solvated electron absorption decayed in two stages: a shorter phase of around 10 ps and a longer phase of more than 3 ns. From a simulation based on a diffusion model we conclude that the diffusion rate of the electron is about 0.8 Ų/ps in wild type PYP, and that the electron is ejected to a short distance of only several angstroms away from the chromophore. The chromophore–protein pocket appears to provide a water-similar local environment for the electron. Because mutations at different places around the chromophore have different effect on the electron recombination dynamics, we suggest that solvated electrons could provide a new method to investigate the local dielectric environment inside PYP and thus help to understand the role of the protein in the photoisomerization process