The flavoprotein WrbA from Escherichia coli is considered to be the prototype of a new family of multimeric flavodoxin-like proteins that are implicated in cell protection against oxidative stress. The present study is aimed at structural characterization of the E. coli protein with respect to its recently revealed oxidoreductase activity. Crystals of WrbA holoprotein in complex with the oxidized flavin cofactor (FMN) were obtained using standard vapour-diffusion techniques. Deep yellow tetragonal crystals obtained from differing crystallization conditions display different space groups and unit-cell parameters. X-ray crystal structures of the WrbA holoprotein have been determined to resolutions of 2.0 and 2.6 A.

• MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• Escherichia coli * metabolismus   MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• represorové proteiny chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• flavinmononukleotid MeSH
• proteiny z Escherichia coli MeSH
• represorové proteiny MeSH
• WrbA protein, E coli MeSH Prohlížeč

Flavin mononucleotide (FMN) belongs to the large family of flavins, ubiquitous yellow-coloured biological chromophores that contain an isoalloxazine ring system. As a cofactor in flavoproteins, it is found in various enzymes and photosensory receptors, like those featuring the light-oxygen-voltage (LOV) domain. The photocycle of FMN is triggered by blue light and proceeds via a cascade of intermediate states. In this work, we have studied isolated FMN in an aqueous solution in order to elucidate the intrinsic electronic and vibrational changes of the chromophore upon excitation. The ultrafast transitions of excited FMN were monitored through the joint use of femtosecond stimulated Raman spectroscopy (FSRS) and transient absorption spectroscopy encompassing a time window between 0 ps and 6 ns with 50 fs time resolution. Global analysis of the obtained transient visible absorption and transient Raman spectra in combination with extensive quantum chemistry calculations identified unambiguously the singlet and triplet FMN populations and addressed solvent dynamics effects. The good agreement between the experimental and theoretical spectra facilitated the assignment of electronic transitions and vibrations. Our results represent the first steps towards more complex experiments aimed at tracking structural changes of FMN embedded in light-inducible proteins upon photoexcitation.

• MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• fotochemické procesy * MeSH
• počítačová simulace MeSH
• Ramanova spektroskopie * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• flavinmononukleotid 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

We present the first report on characterization of the covalent flavinylation site in flavoprotein pyranose 2-oxidase. Pyranose 2-oxidase from the basidiomycete fungus Trametes multicolor, catalyzing C-2/C-3 oxidation of several monosaccharides, shows typical absorption maxima of flavoproteins at 456, 345, and 275 nm. No release of flavin was observed after protein denaturation, indicating covalent attachment of the cofactor. The flavopeptide fragment resulting from tryptic/chymotryptic digestion of the purified enzyme was isolated by anion-exchange and reversed-phase high-performance liquid chromatography. The flavin type, attachment site, and mode of its linkage were determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy of the intact flavopeptide, without its prior enzymatic degradation to the central aminoacyl moiety. Mass spectrometry identified the attached flavin as flavin adenine dinucleotide (FAD). Post-source decay analysis revealed that the flavin is covalently bound to histidine residue in the peptide STHW, consistent with the results of N-terminal amino acid sequencing by Edman degradation. The type of the aminoacyl flavin covalent link was determined by NMR spectroscopy, resulting in the structure 8alpha-(N(3)-histidyl)-FAD.

• MeSH
• Basidiomycota enzymologie   růst a vývoj   metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• flavinadenindinukleotid chemie   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• karbohydrátdehydrogenasy chemie   izolace a purifikace   metabolismus   MeSH
• molekulární konformace MeSH
• nukleární magnetická rezonance biomolekulární metody   MeSH
• oxidace-redukce MeSH
• peptidové fragmenty analýza   MeSH
• sekvenční analýza proteinů metody   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• flavinadenindinukleotid MeSH
• karbohydrátdehydrogenasy MeSH
• peptidové fragmenty MeSH
• pyranose oxidase MeSH Prohlížeč

Flavin mononucleotide (FMN) is a highly efficient photosensitizer (PS) yielding singlet oxygen (1 O2 ). However, its 1 O2 production efficiency significantly decreases upon isoalloxazine ring encapsulation into the protein matrix in genetically encoded photosensitizers (GEPS). Reducing isoalloxazine ring interactions with surrounding amino acids by protein engineering may increase 1 O2 production efficiency GEPS, but at the same time weakened native FMN-protein interactions may cause undesirable FMN dissociation. Here, in contrast, we intentionally induce the FMN release by light-triggered sulfur oxidation of strategically placed cysteines (oxidation-prone amino acids) in the isoalloxazine-binding site due to significantly increased volume of the cysteinyl side residue(s). As a proof of concept, in three variants of the LOV2 domain of Avena sativa (AsLOV2), namely V416C, T418C, and V416C/T418C, the effective 1 O2 production strongly correlated with the efficiency of irradiation-induced FMN dissociation (wild type (WT) < V416C < T418C < V416C/T418C). This alternative approach enables us: (i) to overcome the low 1 O2 production efficiency of flavin-based GEPSs without affecting native isoalloxazine ring-protein interactions and (ii) to utilize AsLOV2, due to its inherent binding propensity to FMN, as a PS vehicle, which is released at a target by light irradiation.

• Klíčová slova
• LOV2 domain, flavin cofactor, genetically encoded photosensitizers, miniSOG, singlet oxygen,
• MeSH
• aminokyseliny MeSH
• flavinmononukleotid chemie   MeSH
• flavoproteiny * chemie   metabolismus   MeSH
• fotosenzibilizující látky * MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• flavinmononukleotid MeSH
• flavoproteiny * MeSH
• fotosenzibilizující látky * MeSH

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in 1O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein.

• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.

• Klíčová slova
• Paracoccus denitrificans, antioxidant enzyme, chromate reductase, flavoprotein, superoxide reductase,
• MeSH
• arginin genetika   MeSH
• flavinmononukleotid chemie   genetika   MeSH
• flaviny genetika   metabolismus   MeSH
• FMN-reduktasa chemie   genetika   MeSH
• katalytická doména genetika   MeSH
• kinetika MeSH
• konformace proteinů * MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce MeSH
• oxidoreduktasy chemie   genetika   MeSH
• Paracoccus denitrificans chemie   enzymologie   MeSH
• sekvence aminokyselin genetika   MeSH
• superoxidy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• arginin MeSH
• chromate reductase MeSH Prohlížeč
• ferric citrate iron reductase MeSH Prohlížeč
• flavinmononukleotid MeSH
• flaviny MeSH
• FMN-reduktasa MeSH
• oxidoreduktasy MeSH
• superoxidy MeSH

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.

• MeSH
• aminokyseliny chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• biokatalýza MeSH
• difrakce rentgenového záření MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• flavoproteiny chemie   genetika   metabolismus   MeSH
• katalytická doména genetika   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• maloúhlový rozptyl MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutageneze cílená MeSH
• NADH, NADPH oxidoreduktasy chemie   klasifikace   metabolismus   MeSH
• NADP chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů * MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• flavinmononukleotid MeSH
• flaviny MeSH
• flavoproteiny MeSH
• NADH, NADPH oxidoreduktasy MeSH
• NADP MeSH

Photosensory receptors containing the flavin-binding light-oxygen-voltage (LOV) domain are modular proteins that fulfil a variety of biological functions ranging from gene expression to phototropism. The LOV photocycle is initiated by blue-light and involves a cascade of intermediate species, including an electronically excited triplet state, that leads to covalent bond formation between the flavin mononucleotide (FMN) chromophore and a nearby cysteine residue. Subsequent conformational changes in the polypeptide chain arise due to the remodelling of the hydrogen bond network in the cofactor binding pocket, whereby a conserved glutamine residue plays a key role in coupling FMN photochemistry with LOV photobiology. Although the dark-to-light transition of LOV photosensors has been previously addressed by spectroscopy and computational approaches, the mechanistic basis of the underlying reactions is still not well understood. Here we present a detailed computational study of three distinct LOV domains: EL222 from Erythrobacter litoralis, AsLOV2 from the second LOV domain of Avena sativa phototropin 1, and RsLOV from Rhodobacter sphaeroides LOV protein. Extended protein-chromophore models containing all known crucial residues involved in the initial steps (femtosecond-to-microsecond) of the photocycle were employed. Energies and rotational barriers were calculated for possible rotamers and tautomers of the critical glutamine side chain, which allowed us to postulate the most energetically favoured glutamine orientation for each LOV domain along the assumed reaction path. In turn, for each evolving species, infrared difference spectra were constructed and compared to experimental EL222 and AsLOV2 transient infrared spectra, the former from original work presented here and the latter from the literature. The good agreement between theory and experiment permitted the assignment of the majority of observed bands, notably the ∼1635 cm-1 transient of the adduct state to the carbonyl of the glutamine side chain after rotation. Moreover, both the energetic and spectroscopic approaches converge in suggesting a facile glutamine flip at the adduct intermediate for EL222 and more so for AsLOV2, while for RsLOV the glutamine keeps its initial configuration. Additionally, the computed infrared shifts of the glutamine and interacting residues could guide experimental research addressing early events of signal transduction in LOV proteins.

• MeSH
• cystein chemie   MeSH
• flavinmononukleotid chemie   MeSH
• fotochemické procesy MeSH
• fototropiny chemie   MeSH
• glutamin chemie   MeSH
• isomerie MeSH
• konformace proteinů MeSH
• molekulární modely MeSH
• normální rozdělení MeSH
• oves chemie   MeSH
• sekvence aminokyselin MeSH
• spektrofotometrie infračervená MeSH
• Sphingomonadaceae chemie   MeSH
• termodynamika MeSH
• vazba proteinů MeSH
• vodíková vazba MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cystein MeSH
• flavinmononukleotid MeSH
• fototropiny MeSH
• glutamin MeSH

The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.

• Klíčová slova
• HPLC, Nqo1, diaphorase, flavodoxin, methionine sulfoxide, thin-layer chromatography,
• MeSH
• difrakce rentgenového záření MeSH
• flavinadenindinukleotid chemie   metabolismus   MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• krystalizace MeSH
• krystalografie rentgenová MeSH
• NAD(P)H dehydrogenasa (chinon) chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• represorové proteiny chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• flavinadenindinukleotid MeSH
• flavinmononukleotid MeSH
• NAD(P)H dehydrogenasa (chinon) MeSH
• proteiny z Escherichia coli MeSH
• represorové proteiny MeSH
• WrbA protein, E coli MeSH Prohlížeč