Real-Time Tracking of Proton Transfer from the Reactive Cysteine to the Flavin Chromophore of a Photosensing Light Oxygen Voltage Protein

Abstract

LOV (light oxygen voltage) proteins are photosensors ubiquitous to all domains of life. A variant of the short LOV protein from Dinoroseobacter shibae (DsLOV) exhibits an exceptionally fast photocycle. We performed time-resolved molecular spectroscopy on DsLOV-M49S and characterized the formation of the thioadduct state with a covalent bond between the reactive cysteine (C72) and C 4a of the FMN. By use of a tunable quantum cascade laser, the weak absorption change of the vibrational band of S−H stretching vibration of C57 was resolved with a time resolution of 10 ns. Deprotonation of C72 proceeded with a time constant of 12 μs which tallies the rise of the thio-adduct state. These results provide valuable information for the mechanistic interpretation of light-induced structural changes in LOV domains, which involves the choreographed sequence of proton transfers, changes in electron density distributions, spin alterations of the latter, and transient bond formation and breakage. Such molecular insight will help develop new optogenetic tools based on flavin photoreceptors.

Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells

“…32 The shift of the absorbance minimum at later time points indicates sample inhomogeneity, possibly due to the presence of two different cysteine rotamers with respect to the S−H torsion as previously observed in other proteins. 28,32,44,45 The peaks at 75 ns and 1 ms of the whole-cell sample were fitted with Gaussians to verify this assumption (Figure S9). The best-fit result was obtained by using two Gaussians with maxima at 2555 and 2549 cm −1 for both time points, supporting the theory of two different cysteine rotamers with different deprotonation dynamics.…”

Capturing the blue-light activated state of the Phot-LOV1 domain fromChlamydomonas reinhardtiiusing time-resolved serial synchrotron crystallography

“…The FTIR difference spectrum in the 1800-1000 cm -1 range was recorded on a Vertex 80V spectrometer (Bruker) in attenuated total reflection (ATR) configuration (Nyquist et al ., 2004), using a diamond ATR cell. For the 2620-2500 cm -1 range, the sample was sandwiched and sealed between two BaF 2 windows and difference spectra were taken in transmission mode (Maia et al ., 2021). In both configurations, crystals in mother liquor at pH 6.5 were kept in the dark for 300 s, followed by 10 s of illumination with a LED emitting at a center wavelength of 450 nm (∼10 mW cm -2 ).…”

Genetically encoded non‐canonical amino acids reveal asynchronous dark reversion of chromophore, backbone, and side‐chains in EL222

“…Non‐irradiated LOV proteins absorb maximally at ~450 nm while their irradiated counterparts peak at ~390 nm (Figure 1b left panel). This frequency shift is caused by photon absorption by the oxidized FMN cofactor, which triggers its partial reduction to a semiquinone radical and adduct formation with a conserved cysteine residue via the C4A atom (see FMN atom numbering in Figure S2a) (Maia et al 2021). FMN‐Cys covalent bond formation takes place in the nanosecond to microsecond time scale (Iuliano et al 2018; Andrikopoulos et al 2021; Liu et al 2023).…”