The PHOT1 (NPH1) gene from Avena sativa specifies the blue light receptor for phototropism, phototropin, which comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain. Light exposure is conducive to autophosphorylation of the protein kinase domain. We have reconstituted a recombinant LOV2 domain of A. sativa phototropin with various 13 C͞ 15 N-labeled isotopomers of the cofactor, FMN. The reconstituted protein samples were analyzed by NMR spectroscopy under dark and light conditions. Blue light irradiation is shown to result in the addition of a thiol group (cysteine 450) to the 4a position of the FMN chromophore. The adduct reverts spontaneously in the dark by elimination. The light-driven flavin adduct formation results in conformational modification, which was diagnosed by 1 H and 31 P NMR spectroscopy. This conformational change is proposed to initiate the transmission of the light signal via conformational modulation of the protein kinase domain conducive to autophosphorylation of NPH1. N umerous phenomena in the life cycle of plants, including phototropism, stomatal opening, and circadian periodicity, are modulated by light. Photoreceptors responsive to UV, blue, red, and far red light have been reported. Together, they span the spectral range of about 280-800 nm.Blue light-responsive processes have been known for a long time, but cognate receptors have been characterized only recently. Thus, cryptochromes characterized by sequence similarity to DNA photolyases are now believed to be involved in the synchronization of the circadian clock (1-3). Phototropins are involved in phototropism (4, 5) and have no sequence similarity with the cryptochrome group. FAD and FMN serve as chromophores for cryptochromes and phototropins, respectively (6).Phototropin of Avena sativa is a protein with 923 amino acids specified by the NPH1 gene (ref. 7; for review, see also ref. 8).The protein comprises two FMN-binding LOV domains and a serine͞threonine protein kinase domain.The LOV domains of phototropin are similar to PAS domains involved in light, oxygen, or voltage sensing (4) in a variety of regulatory as well as sensor proteins exhibiting diverse functions (4, 9, 10). Recombinant LOV1 and LOV2 domains of phototropins from different plants have been shown to bind FMN (11). The crystal structure of the LOV2 domain of PHY3 protein of Adiantum capillus-veneris has been published recently (12).In this study, we reconstituted the LOV2 apoprotein from A. sativa with Experimental ProceduresMaterials. Stable isotope-labeled FMN samples were prepared by published procedures (13-15). Recombinant LOV2 Domain of Phototropin.A recombinant fusion protein comprising the calmodulin-binding domain from myosin light chain kinase and the LOV2 domain from NPH1 protein of A. sativa was prepared as described earlier (16,17). The fusion protein is subsequently designated recombinant LOV2 domain.Reconstitution of LOV2 Domain with Isotope-Labeled FMN. Recombinant LOV2 domain (fusion protein) was depleted of FMN and su...
Several chemically substituted flavins are investigated in the oxidized and the reduced state by 13C and 15N NMR techniques. The dependence on the polarity of the solvent and on the concentration is studied. In combination with already published results, a semiempirical theory is developed to interpret the chemical shifts in terms of the solution structure of flavins. Where possible, the results are compared with crystallographic and light absorption data. In contrast to common ideas, the solution structure of the oxidized state is not fully coplanar, but the N(10) atom is situated out of plane to a certain degree. Polarizing the flavin by hydrogen bonds in a high dielectric medium moves the N(10) atom into the molecular plane, and the flavin molecule becomes coplanar. In the coplanar molecule, pi electrons are delocalized from the N(10) atom mainly to O(2 alpha) and O(4 alpha). The NMR results show that the solution structure of reduced flavin is mainly governed by sterical hindrance and hydrogen bonds. The findings are in contrast to commonly accepted ideas that reduced flavin is strongly bent. In an apolar solvent, the reduced neutral isoalloxazine is only slightly bent. The formation of hydrogen bonds in a protic solvent of a high dielectric constant decreases the bend. The N(10) atom is now almost fully sp2 hybridized, and the N(5) atom has an endocyclic angle of 115-117 degrees, indicating its predominant sp2 character. The results have several important implications for flavin catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)
Partially deuterated and various substituted flavin and thiaflavin model compounds have been synthesized. For the first time, high-resolution H, D, and 14N ENDOR and TRIPLE resonance experiments in fluid solutions have been performed on the paramagnetic derivatives of these compounds. Additionally, valuable information has been obtained about hyperfine anisotropies and molecular structures from ENDOR in rigid matrices. Solid matrix ENDOR studies of native flavoenzymes, namely, "Old Yellow Enzyme" (NADPH dehydrogenase), two flavodoxins, and a methanol oxidase are reported. The ENDOR matrix signals of the various flavoproteins are different in intensity, suggesting that the microenvironments are remarkably different. Applicabilities and limitations of the ENDOR technique in the studies of flavins and flavoenzymes are discussed.
The dependence on pH of the disproportionation of the flavosemiquinone has been investigated by ESR. For 3-alkylated lumiflavin [R(3) in aqueous solution p i (for the dissociation of the neutral flavosemiquinone) is 8.4; PKred is 6.3. Hydrolytic ring cleavage in position 2, 8,8'-dimerization, and 10-dealkylation interfere with the formation of the flavosemiquinone anion. However, in non-aqueous dimethylformamide and with stoichiometric amounts of strong base ( t -B u O K ) , 100 Olio flavosemiquinone anion is formed without interfering side reactions. Its light absorption spectrum is practically identical to that of the "red flavoprotein radical".From the ESR spectra the individual isotropic hyperfine coupling constants have been determined.Since the discovery of flavin radicals by Michaeljs and co-workers [1,2] there has been doubt as to the correlation of visible absorption spectra and protonation state of the flavosemiquinone. Beinert [3] was the first to interpret the light absorption spectra of the half-reduced flavin system as a function of pH. I n a previous paper [4] we have made further correlations between structure, light absorption and other physical properties of flavin species.Within the range of these previous investigations two essential aspects remained : (a) determination of the light absorption of the flavosemiquinone anion, and (b) investigation of the tautomer structure of the neutral flavosemiquinone that absorbs in the range of 560-600 nm, as revealed by Beinert [3].The biochemical importance of these two points is stressed by Massey and Palmer [5] who found two classes of radical intermediates at half reduction of flavoproteins. The first type of radical, which shows red colour, has been found e.g. in D-amino acidoxidase, glucose oxidase a t high pH, oxynitrilase, and L-amino acid oxidase. The other type, the blue flavin radical, was found in the Azotobacter vinelandii flavoprotein, glucose oxidase a t low pH, and thioredoxin reductase. To be sure, these radicals, which are quantitatively verified by electron spin resonance (ESR), must not be confused with other coloured intermediates that have Non-Standard Abbreviations. Neutral flavoquinone, Floo,R(I) ; neutral flavosemiquinone, F1RH ; neutral flavohydroquinone,
We report a detailed spectroscopic investigation of the chiral molecule bromochlorofluoromethane (CHBrClF) with rotational resolution using a pulsed nozzle beam Fourier transform microwave (FTMW) and a waveguide FTMW spectrometer as well as a supersonic jet interferometric Fourier transform infrared (FTIR) and infrared diode laser spectrometer. The rotational spectrum of CHBrClF has been measured between 8 and 18 GHz. The quadrupole hyperfine components have been fully resolved for the assigned rotational transitions with J⩽18. Three ground state rotational constants, five centrifugal distortion constants, and all five independent elements of the bromine and chlorine quadrupole coupling tensors have been determined for each of the four isotopomers CH79Br35CIF, CH81Br35CIF, CH79Br37CIF, and CH81Br37CIF from about 500 measured transition frequencies of the hyperfine components. The quadrupole coupling tensor has been transformed to its principal axes. The determinable sign combinations of the off-diagonal elements of the coupling tensor have been evaluated. Rotational transitions involving high J were measured by FTIR spectroscopy between 15 and 40 cm−1 (450–1200 GHz) using a light pipe cell, providing an estimate of the permanent dipole moment |μ|=(1.5±0.3) D from intensities. In the midinfrared, we have fully analyzed the rovibrational line structure of supersonic jet spectra of the CF-stretching fundamental ν4, giving band centers for the isotopomers CH79Br35CIF [ν̃ 40=1077.178 43(4) cm−1], CH81Br35CIF [ν̃=1077.133 06(4) cm−1], CH79Br37CIF [ν̃ 40=1076.7914(4) cm−1], and CH81Br37CIF [ν̃ 40=1076.730 26(5) cm−1]. A combined analysis of about 20 microwave frequencies, more than 100 infrared ground state combination differences, and about 70 infrared transition frequencies for each of the35Cl isotopomers finally provide accurate ground and excited state rotational parameters as well as structural parameters, which may be compared to ab initio calculations. The results are discussed in relation to the molecular structure as well as coincidences of ν4 absorptions with CO2 laser lines in view of CO2–laser pumping and possible spectroscopic studies of this chiral molecule at ultrahigh resolution.
The neutral flavosemiquinone has been studied in detail by electron spin resonance spectroscopy. Isotopic (W, 2H) and chemical substitutions have been carried out. A hyperhe coupling scheme of the neutral flavosemiquinone is described where N(5), N(10), CH,(10), CH3(8) and H(6) are involved. The highest spin density is located a t N(5) as has also been found for the anionic and chelated form of the flavosemiquinone. The exchangeable proton in the neutral flavosemiquinone is attached to N(5) having a coupling constant of 7.6 G which is about the same magnitude as the coupling constant to N(5). The hyperfine couplings are interpreted in terms of spin densities and comparison is made with available quantum chemical calculations. Evidence is presented that two tautomeric forms of the neutral flavosemiquinone have to be considered which can be obtained in a pure form by suitable substitution of flavin derivatives. The two types of neutral flavosemiquinones are easily distinguishable by their electron spin resonance and light absorptions.Since the discovery of flavin radicals by Michaelis and co-workers [l] it has been tacitly assumed that the addition of a hydrogen atom to flavoquinone (I) would occur a t N(1) yielding a neutral flavin radical PlRH of structure 11. This was in analogy to the (in the meantime well established) protonation of flavoquinone, yielding the flavoquinone cation 111 [2]. I1 B~R H IIaThis idea was also supported by the fact that PlRH showed an acidity comparable to that of flavohydroquinone (IV) as established by Michaelis and Schwarzenbach potentiometrically [3]. These authors found pKglBE g 7 as compared to pKfireaRH, = 6.5. Clearly the most acidic proton in IV (R = H) must be assumed to dissociate from the cyclic imide group in either position 1 or 3, and not from the pyrrole" nitrogen in position 5 of the isoalloxazine ring. The validity of this assumption for l75-dihydroflavins was proved by the fact that 5,10-dihydro-l,3-dimethyllumichrome (V) does not exhibit a measurable acidity. Nomenclature. The term flavin means the 10-alkylated 6,7-benzo-pteridine-2,4-dione, i. e. the isoalloxazine nucleus, irrespective of the redox state. Its oxidized form is called "flavoquinone", its fully reduced form "flavohydroquinone", 1,5-dihydroflavin or, for convenience, leucoflavin, though the free leucoflavin is, in fact, not colorless. The term "flavosemiquinone" is assigned to the intermediate radical form. Lumiflavin means 7,8,10-trimethylflavin.' <
Flavin derivatives, enriched with 5N (w 95 %) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two-electron reduced state by the 5N nuclear magnetic resonance technique. The measurements were conducted with aqueous and chloroform solutions of flavin. A comparison of the chemical shifts of the N(l) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen-bridge formation to these atoms. The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction.A pK, of 6.8 was determined from pH-dependent 15N NMR measurements of the two-electron reduced flavin molecule. In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution. The 'N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein. Especially the N(l) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein. In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure. These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium MP.Where possible 15N-'H, 15N-15N and 13C-15N coupling constants were determined. Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein-bound flavin and for the evaluation of the interaction between flavin and apoprotein.Spin-lattice relaxation measurements show that the relaxation of the 5N(3)H group of flavin is predominantly determined by dipole-dipole interaction. The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.Flavodoxins are proteins of small molecular mass (= 15000 Da) functioning as electron carriers in low-potential oxidation-reduction reactions [l]. The prosthetic group of flavodoxins is riboflavin 5'-phosphate (FMN). The flavodoxins contain no metal ions but are able to replace the ironsulfur proteins ferredoxins in biological reactions.The three-dimensional structure of the Clostridium MP flavodoxin is known [l]. The crystal structure of the more easily available, related flavodoxin from Megasphaera elsdenii is not known, but its physical and chemical properties have been studied in detail [l]. The NMR technique is a valuable tool to study the specific interaction between the apoprotein and the prosthetic group. It has been postulated that such specific interactions are responsible for the specific biological reaction catalyzed by a particular flavoprotein [2]. In this respect the 'H and 13C NMR technique has already been applied succesfully to M . elsdenii flavodoxin [3, 41.Abbreviations. FMN, riboflavin 5'-phosphate; FMNH, and FMNH-, neutral and anionic l,Sdihydro-FMN, respectively; ...
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