<sup>13</sup>C‐NMR. Study on Isoalloxazine and Alloxazine Derivatives

Grande, Hans J.; Gast, R. G.; Schagen, C.G. van; Berkel, Willem J.H. van; Müller, Franz

doi:10.1002/hlca.19770600208

Cited by 48 publications

(29 citation statements)

References 14 publications

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“…These tensor elements, i.e., Q, , , dry and ozzr are at 75 ppni, 104 ppni and 217 ppm. Calculation of the average chemical shift oav'(Eqn 4) gives 132 ppm which is in good agreement with the value of 136 ppm observed in the liquid state [8,38]. Using Eqns (2) and ( 3 ) , do and are calculated as 127 ppm and 0.34, respectively.…”

Section: Solirl-state N M R Of Flavinsupporting

confidence: 76%

“…This was achieved experimentally by varying the repetition time between 1 h and 10 s. In this way it is possible to estimate roughly the TI values: the peaks at 20 ppm (peak 6), 60 ppm (peak 5 ) and 120 ppm (peak 2) have relatively short TI values. In liquidstate spectra of flavins [38] the C7 and C8 carbon atoms, the C atoms of the ribityl side chain and the C6 and C9 atoms are found respectively at these frequencies. The peaks 1, 3 and 4 in Fig.…”

Section: Solirl-state N M R Of Flavinmentioning

confidence: 90%

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On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

Moonen

Müller

1983

European Journal of Biochemistry

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The mobility of the isoalloxazine ring of the prosthetic group of Megaspliuera clsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (> 90 ' %, 13C) prosthetic group was bound to the apoprotein. T, and Tz values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are i n agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent Tl and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall Ti values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium M P .In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.Internal motions in proteins have recently attracted considerable intercst since the realisation that such motions play an important role in cooperative and allosteric effects. Moreover, the internal mobility in enzymes probably contributes to the catalytic effects of biomolecules [I]. Theoretical calculations by Karplus and McCaiiiinon [2,3] indicate that such motions can occur on the nanosecond -picosecond time scale despite the close-packed structure of native proteins. Such an internal mobility in the nanosecond time range has recently been demonstrated in the flavoprotein lipoamide dehydrogenase [4].Flavoproteins occur widely in nature and catalyze a variety of biological reactions. The physical and chemical properties of the flavin prosthetic group are perturbed upon binding to a particular apoflavoprotein. These observations and model studies led to the postulation [5] that the molecular basis for the specificity of a particular flavoprotein could be a specific interaction between the apoprotein and the prosthetic group; i.e. hydrogen bond formation between some amino acid residues and certain atoms of the prosthetic group. That such hydrogen bonds do influence the orbital structure of the flavin molecule has recently been demonstrated by Eweg et al. [6]. both by photoelectron spectroscopy and from theoretical calculati...

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Section: Solirl-state N M R Of Flavinsupporting

confidence: 76%

Section: Solirl-state N M R Of Flavinmentioning

confidence: 90%

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

Moonen

Müller

1983

European Journal of Biochemistry

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“…As will be shown below, the formation of a hydrogen bond between N(l) of flavin and water molecules cannot be excluded as a possible source for the observed, peculiar relaxation behavior of the 15N(l) atom of flavin. On the other hand, although the CHC1, used was carefully freed from water prior to use, we had to add a small amount of methanol to the flavin solution (to prevent a slow undesired reaction) [17]. This probably explains our observation and that of Yagi et al [14] and Kawano et al [15].…”

Section: N1101mentioning

confidence: 83%

Nuclear‐magnetic‐resonance investigation of ¹⁵N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin

Franken

Rüterjans

Müller

1984

European Journal of Biochemistry

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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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“…Aliquots of concentrated [4a-13C]FMN and gaseous oxygen were injected through a septum cap. (13) in 85% yield. The phosphorylation of the riboflavin was carried out by a described method (14), adapted to small scale, with average yield of 60-65% of the crude phosphorylation product.…”

mentioning

confidence: 98%

Structure of the oxygen adduct intermediate in the bacterial luciferase reaction: ¹³ C nuclear magnetic resonance determination

Ghisla

Hastings

Favaudon

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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By using FMN enriched in '3C (90%) at position C-4a, we have conclusively shown that the reaction of molecular oxygen with bacterial luciferase-bound FMNH2 forms an adduct at the 4a position. Consistent with this are 13C NMR studies of FMN and other flavin compounds which show that this carbon should be unusually reactive in the reduced 1,5-dihydroflavins with respect to electrophilic attacks. An intermediate of the bacterial luciferase reaction, formed upon the addition of molecular oxygen to the reduced flavin mononucleotide/enzyme complex, has been isolated by chromatography at low temperature and characterized spectrally (1, 2). Spectrally related transient intermediates have also been observed in certain flavin-dependent hydroxylases (3). Absorption spectra studies of the oxidation of free 1,5-dihydroflavins by oxygen in aqueous and aprotic media have also given clear indication of the existence of a flavin/oxygen adduct (4, 5). Recent evidence (3, 6) strongly suggests a flavin C-4a peroxide structure for these adducts; however, in view of the great variability of the spectra of reduced flavins depending on substituents and environment (6, 7), the absorption spectra of the intermediates cannot be considered to be sufficiently diagnostic. The structures of these oxygen adducts are, thus still disputed, and those of certain other intermediates implicated in the reaction of flavoprotein oxidases are even more speculative (8).Although the 4a-carbon is now generally considered as the best candidate for the primary electrophilic addition of molecular oxygen, other positions, including C-6, C-8, C-9a, and C-10a, are still discussed (9, 10). The rearrangement of such primary adducts governed by apoprotein conformational changes has also been proposed (9). In this paper we present direct evidence for addition of oxygen at C-4a in the reduced flavin complexed to luciferase by using selectively enriched FMN measured by 13C NMR. These experiments offered also the opportunity to investigate the conditions of application of 13C NMR for the analysis of enzymatic intermediates stabilized at subzero temperatures.MATERIALS AND METHODS The luciferase was isolated from the luminous bacterium Beneckea harveyi, mutant strain M-17 (11), and purified according to Baldwin et al. (12). Samples for the NMR experiments were prepared in mixtures of 20% ethylene glycol-d4 and phosphate buffer (0.3 M, pH 7.0), which permitted cooling to -20'C without freezing. The concentration of luciferase, measured by absorption using an e-2m value of 74 mM'1 cm-l for the heterodimer (12), was in the range of 1.1-1.6 mM. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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¹³C‐NMR. Study on Isoalloxazine and Alloxazine Derivatives

Cited by 48 publications

References 14 publications

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

Nuclear‐magnetic‐resonance investigation of ¹⁵N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin

Structure of the oxygen adduct intermediate in the bacterial luciferase reaction: ¹³ C nuclear magnetic resonance determination

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13C‐NMR. Study on Isoalloxazine and Alloxazine Derivatives

Cited by 48 publications

References 14 publications

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by 13C‐Nuclear‐Magnetic‐Resonance Relaxation

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by 13C‐Nuclear‐Magnetic‐Resonance Relaxation

Nuclear‐magnetic‐resonance investigation of 15N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin

Structure of the oxygen adduct intermediate in the bacterial luciferase reaction: 13 C nuclear magnetic resonance determination

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¹³C‐NMR. Study on Isoalloxazine and Alloxazine Derivatives

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by ¹³C‐Nuclear‐Magnetic‐Resonance Relaxation

Nuclear‐magnetic‐resonance investigation of ¹⁵N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin

Structure of the oxygen adduct intermediate in the bacterial luciferase reaction: ¹³ C nuclear magnetic resonance determination