Red Shift in the Absorption Spectrum of Phototropin LOV1 upon the Formation of a Semiquinone Radical: Reconstructing the Orbital Architecture

Abstract

Flavin mononucleotide (FMN) is a ubiquitous blue-light pigment due to its ability to drive one- and two-electron transfer reactions. In both light-oxygen-voltage (LOV) domains of phototropin from the green algae Chlamydomonas reinhardtii, FMN is noncovalently bound. In the LOV1 cysteine-to-serine mutant (C57S), light-induced electron transfer from a nearby tryptophan occurs, and a transient spin-correlated radical pair (SCRP) is formed. Within this photocycle, nuclear hyperpolarization is created by the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect. In a side reaction, a stable protonated semiquinone radical (FMNH^·) forms undergoing a significant bathochromic shift of the first electronic transition from 445 to 591 nm. The incorporation of phototropin LOV1-C57S into an amorphous trehalose matrix, stabilizing the radical, allows for application of various magnetic resonance experiments at ambient temperatures, which are combined with quantum-chemical calculations. As a result, the bathochromic shift of the first absorption band is explained by lifting the degeneracy of the molecular orbital energy levels for electrons with alpha and beta spins in FMNH^· due to the additional electron.

Figure 1

(A) Chemical structure of FMN (F_L, yellow) with its relevant atom positions, with R representing the ribityl side chain. Reduction and subsequent protonation lead to a semiquinone radical (FMNH^·, turquois). Shown below is the structure of tryptophan with its one-letter code W. (B) Simplified photocycle of phototropin LOV1 C57S from C. reinhardtii. Light excitation forms the singlet excited state of F_L which undergoes intersystem crossing (ISC) to a long-lived molecular triplet. Subsequent electron transfer from tryptophan W98 forms a spin-correlated radical pair (SCRP) coherently oscillating coherently between the electronic triplet and singlet states inducing photo-CIDNP nuclear hyperpolarization which can be detected with NMR. Furthermore, byproduct FMNH^· is formed by a side reaction quitting the photo-CIDNP photocycle.

Figure 2

UV/vis absorption spectrum of phototropin LOV1 C57S in a glassy trehalose matrix on a Petri dish. The signal background of the Petri dish was removed by a blank measurement. Measurements were conducted at the same spot with the flavin in its oxidized form (yellow) and after 488 nm irradiation with a cw-laser (green) forming FMNH^·. At shorter wavelengths, the scattering background is clearly visible. Excitation energies and oscillator strengths for the brighter transitions from quantum chemical calculations are shown as dotted sticks, and the inset indicates the two kinds of states.

Figure 3

¹⁵N solid-state photo-CIDNP spectra of u-[¹⁵N] CrLOV1 C57S in a trehalose matrix at room temperature (red, top) and in a frozen solution at 235 K (green, bottom) at 4.7 T (200 MHz ¹H frequency). Three light-induced signals are observed at 342 ppm (assigned to FMN-N5), 156 ppm (FMN-N10), and 129 ppm (W98-N1).

Figure 4

Cross sections (solid lines) from ¹⁵N photo-CIDNP SUPER MAS NMR experiments in trehalose glass (red, top) and in the frozen solution (green bottom) for (A) FMN-N5 (δ_iso = 342 ppm), (B) FMN-N10 (δ_iso = 156 ppm), and (C) W98-N1 (δ_iso = 129 ppm). Fit curves obtained from simulations are shown as dotted lines. The principal values (δ₁₁, δ₂₂, δ₃₃) as well as the span (Ω [ppm]) and the asymmetry factor (η) are extracted from fitting and displayed in Table S3.

Figure 5

X- (A) and Q-band (B) CW EPR spectra of CrLOV1 at room temperature after the formation of FMNH^·. The experimentally determined g-factor is 2.0039. The simulated fit curve is shown in red, taking into account g = [2.0044 2.0038 2.0019]⁴ and the HFCs of N5, N10, and H5 shown in Table 2.

Figure 6

X-band (A) and Q-band (B) experimental ¹H Davies ENDOR spectra of FMNH^· at 90 K (black line). The fitting curves result from HFC from the assigned protons of FMN (H1′ (green), H5 (purple), H-6 (orange), and H8α (blue) as well as from small HFC contributions (red) related to H7α and H9 and the surrounding protein matrix. HFC data are presented in Table 2.

Figure 7

Scheme of frontier molecular orbitals for FMN and FMNH^·. In the case of the latter, orbitals for the two spins are shown separately. For FMN, the lowest electronic transition corresponds to the excitation of an electron from HOMO to LUMO (S₁), whereas the second intense transition is an excitation from HOMO-1 to LUMO (S₄). All three molecular orbitals involved exhibit π character; see also Figure S2 with the depiction of the relevant MOs. In case of FMNH^·, energy levels for the two spin states of the same orbital are shown separately. The first two transitions with oscillator strengths larger than 0.05 (D₁, D₃) are similar in nature to the ones in FMN, as the involved orbitals for the β spin resemble the orbitals for the closed shell FMN (S₁, S₄). In contrast to this, the third transition (D₅) is dominated by the excitation of an electron with α spin from SOMO to LUMO+2.