Perturbation of the ground-state electronic structure of FMN by the conserved cysteine in phototropin LOV2 domains

Abstract

In LOV2, the blue-light sensitive domain of phototropin, the primary photophysical event involves intersystem crossing (ISC) from the singlet-excited state to the triplet state. The ISC rate is enhanced in LOV2 as compared to flavin mononucleotide (FMN) in solution, which likely results from a heavy-atom effect of a nearby conserved cysteine, C450. Here, we applied fluorescence line narrowing (FLN), resonance Raman (RR) and Fourier-transform infrared (FTIR) spectroscopy to investigate the electronic structure of FMN bound to Avena sativa LOV2 (AsLOV2), its C450A mutant and Adiantum LOV2 (Phy3LOV2). We demonstrate that FLN is the method of choice to obtain accurate vibrational spectra on highly fluorescent flavoproteins. The vibrational spectrum of AsLOV2-C450A showed small but significant shifts with respect to those of wild type AsLOV2 and Phy3LOV2, with a systematic down-shift of Ring I vibrations, upshifts of Ring II and III vibrations and an upshift of the C2=O mode. These trends are similar to those in FMN model systems with an electron-donating group substituted at Ring I, known to induce a quinoid character to the electronic structure of oxidized flavin. Thus, enhancement of the ISC rate in LOV2 is induced through weak electron donation by the cysteine which mixes the FMN pi-electrons with the heavy sulfur orbitals, manifesting itself in a quinoid character of the ground electronic state of oxidized FMN. The proximity of the cysteine to FMN thus not only enables formation of a covalent adduct between FMN and cysteine, but also facilitates the rapid electronic formation of the reactive FMN triplet state.

Effect of computational methodology on the conformational dynamics of the protein photosensor LOV1 from Chlamydomonas reinhardtii

“…Moreover, they proposed that it increases the intersystem crossing (ISC) rate by rapid generation of the reactive FMN triplet state, leading to a shortening of the FMN singlet excitedstate lifetime. These conclusions are consistent with several experimental studies, which state that the presence of the reactive cysteine at about 4 Å of the FMN [12,68,69] is responsible for a shortening of the singlet excited state lifetime and increase of the ISC rate by a factor of 2-3 as compared to FMN in solution [63,70] or cysteine-less mutants [71,72]. In this context, it is worth pointing out that in LOV domains an efficient ISC rate is crucial.…”

“…2S of the "Electronic supplementary material". They concluded this by comparing vibrational spectra from fluorescence line narrowing experiments of the wild-type LOV2 species AsLOV2 and Phy3LOV2 with a cysteine-less mutant AsLOV2-C450A [63]. They found that the mutant shows small but significant frequency shifts of the FMN ring vibrations as well as of the C2=O mode with respect to the wild-type species, similar to those found in FMN model systems with an electron-donating group substituted at the C8 position of the FMN.…”

“…In our simulation study, we find that the FMN-N5-atom stays H-bonded with the reactive Cys57 residue in the time range before the structural rearrangement, while the H-bond gets disrupted in the time range after the re-arrangement. Kennis et al [63] suggested in the case of LOV2 that the presence of the thiol group of Fig. 6 Representative configurations showing the orientations of the Cys57-thiol group with regard to the FMN ring a before and b after the structural re-arrangement, obtained with the non-invasive PME technique the reactive cysteine residue in vicinity of the aromatic ring of FMN shifts the resonance equilibrium away from the oxidized form of FMN to one of its quinoid forms, which is depicted in Fig.…”

Mechanism-based tuning of a LOV domain photoreceptor

“…Met-to-aliphatic side chain substitutions at analogous positions in flavodoxins affect the stability of reduced flavin species28. In addition, the presence of sulphur atoms in LOV domain active sites has been shown to impart quinoid character to the flavin18,29. Thus, substitutions of Met135 and Met165 in VVD should alter the electronics of the flavin ring and affect adduct stability.…”

Post-translational control of protein function with light using a LOV-intein fusion protein

“…20 The level of LOV-independent splicing, and the effect of blue light on the splicing of Npu inteins was measured using a C450S mutant of LOV2 that has previously been shown to be locked in its dark state even in the presence of blue light. 23 We observed no difference in spliced extein levels in samples incubated in the dark or in the presence of blue light ( Figure 2C), indicating that a functional LOV2 domain is required for the regulation InN-LOV-∆6InC splicing with blue light. Interestingly, incorporation of LOV C450S into the original Kan RInN-LOV-InC complex had no affect on the relative levels of spliced extein (Supplementary Figure 7), further indicating that the high affinity of the Npu split-intein fragments overrides any conformational inhibition of splicing when LOV is in its dark state.…”