Sub-Millisecond Photoinduced Dynamics of Free and EL222-Bound FMN by Stimulated Raman and Visible Absorption Spectroscopies

Abstract

Time-resolved femtosecond-stimulated Raman spectroscopy (FSRS) provides valuable information on the structural dynamics of biomolecules. However, FSRS has been applied mainly up to the nanoseconds regime and above 700 cm−1, which covers only part of the spectrum of biologically relevant time scales and Raman shifts. Here we report on a broadband (~200–2200 cm−1) dual transient visible absorption (visTA)/FSRS set-up that can accommodate time delays from a few femtoseconds to several hundreds of microseconds after illumination with an actinic pump. The extended time scale and wavenumber range allowed us to monitor the complete excited-state dynamics of the biological chromophore flavin mononucleotide (FMN), both free in solution and embedded in two variants of the bacterial light-oxygen-voltage (LOV) photoreceptor EL222. The observed lifetimes and intermediate states (singlet, triplet, and adduct) are in agreement with previous time-resolved infrared spectroscopy experiments. Importantly, we found evidence for additional dynamical events, particularly upon analysis of the low-frequency Raman region below 1000 cm−1. We show that fs-to-sub-ms visTA/FSRS with a broad wavenumber range is a useful tool to characterize short-lived conformationally excited states in flavoproteins and potentially other light-responsive proteins.

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

“…The dark‐to‐lit transition of EL222 measured by time‐resolved vibrational spectroscopy is typically analyzed using a sequential kinetic model. A singlet species ( 1 FMN*) is nearly instantaneously formed upon photon absorption, followed by the triplet species ( 3 FMN*) in ~3 ns, and finally the adduct state (A 390 ) in ~5 μs (or ~600 ns in H 2 O‐based buffer) (Iuliano et al 2018; Andrikopoulos et al 2021; Liu et al 2023). Because the steady‐state difference spectra are different from the transient spectra of the adduct state obtained a few microseconds after irradiation (Figure S25), the existence of an additional intermediate species in the multi‐millisecond time scale is envisioned, as previously postulated (Iuliano et al 2018).…”

“…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). The rupture of such an adduct state, sometimes referred to as A 390 species (Iuliano et al 2018), is much slower, typically returning to the resting state in seconds to hours, depending on the particular LOV domain (Lebedev et al 2014).…”

On the feasibility of time-resolved X-ray powder diffraction of macromolecules using laser-driven ultrafast X-ray sources

“…Using one laser in a conventional splitand-delay setup, a timing jitter between pump and probe pulses of a few fs can be routinely achieved. Alternatively, by using two laser amplifiers seeded by the same oscillator, time delays up to a full ms between amplifier pulses can be achieved, still with fs precision, as has been verified in optical spectroscopy measurements (Liu et al, 2023).…”