Flavocytochrome c sulfide dehydrogenase (FCC) is an important enzyme of sulfur metabolism in sulfur-oxidizing bacteria, and its catalytic properties have been extensively studied. However, the ultrafast dynamics of FCC is not well understood. We present ultrafast transient absorption and fluorescence spectroscopy measurements to unravel the early events upon excitation of the heme and flavin chromophores embedded in the flavocytochrome c (FccAB) from the bacterium Thiocapsa roseopersicina. The fluorescence kinetics of FccAB suggests that the majority of the photoexcited species decay nonradiatively within the first few picoseconds. Transient absorption spectroscopy supports these findings by suggesting two major dynamic processes in FccAB, internal conversion occurring in about 400 fs and the vibrational cooling occurring in about 4 ps, mostly affecting the heme moiety.

• Publikační typ
• časopisecké články MeSH

The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny * chemie   metabolismus   genetika   MeSH
• DNA vazebné proteiny * chemie   metabolismus   MeSH
• DNA * metabolismus   chemie   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny * chemie   metabolismus   MeSH
• fotoreceptory mikroorganismů * chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů účinky záření   MeSH
• krystalografie rentgenová MeSH
• molekulární modely MeSH
• oxidace-redukce MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• transkripční faktory * chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny * MeSH
• DNA vazebné proteiny * MeSH
• DNA * MeSH
• flavinmononukleotid * MeSH
• flaviny * MeSH
• fotoreceptory mikroorganismů * MeSH
• transkripční faktory * MeSH

Photoreceptors containing the light-oxygen-voltage (LOV) domain elicit biological responses upon excitation of their flavin mononucleotide (FMN) chromophore by blue light. The mechanism and kinetics of dark-state recovery are not well understood. Here we incorporated the non-canonical amino acid p-cyanophenylalanine (CNF) by genetic code expansion technology at 45 positions of the bacterial transcription factor EL222. Screening of light-induced changes in infrared (IR) absorption frequency, electric field and hydration of the nitrile groups identified residues CNF31 and CNF35 as reporters of monomer/oligomer and caged/decaged equilibria, respectively. Time-resolved multi-probe UV/visible and IR spectroscopy experiments of the lit-to-dark transition revealed four dynamical events. Predominantly, rearrangements around the A'α helix interface (CNF31 and CNF35) precede FMN-cysteinyl adduct scission, folding of α-helices (amide bands), and relaxation of residue CNF151. This study illustrates the importance of characterizing all parts of a protein and suggests a key role for the N-terminal A'α extension of the LOV domain in controlling EL222 photocycle length.

• Klíčová slova
• FTIR spectroscopy, UV/vis spectroscopy, flavoproteins, genetic code expansion, kinetics, photosensory receptors, protein structural dynamics, signal transduction, site-specific vibrational probes, time-resolved methods,
• MeSH
• aminokyseliny * metabolismus   MeSH
• flavinmononukleotid * chemie   MeSH
• regulace genové exprese MeSH
• transkripční faktory metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny * MeSH
• flavinmononukleotid * MeSH
• transkripční faktory MeSH

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.

• Klíčová slova
• femtosecond-stimulated Raman spectroscopy (FSRS), flavins, kinetic isotope effect (KIE), lifetime distribution analysis (LDA), light-oxygen-voltage (LOV) photosensors, maximum entropy method, photobiology, photochemistry, protein structural dynamics, time-resolved vibrational spectroscopy, transient visible absorption (visTA) spectroscopy,
• MeSH
• Ramanova spektroskopie * metody   MeSH
• spektrofotometrie infračervená MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH