Affordable Access

The application of various NMR techniques to free and protein-bound flavins : an approach to elucidate the active center of flavoproteins

  • van Schagen, C.G.
Publication Date
Jan 01, 1983
Wageningen University and Researchcenter Publications
External links


The subject of this thesis is the application of high resolution NMR techniques to study the structure of free and protein-bound flavins. The main part of the thesis deals with low molecular weight flavoproteins, especially with the flavodoxins from M.elsdenii and A.vinelandii. The model studies served as a basis for the interpretation of the corresponding results obtained with flavoproteins.Chapter 2 describes the 13 C NMR properties of the two-electron reduced free flavins in apolar solvents. It was found that a correlation exists between the calculated and observed 13 C chemical shifts. This correlation relates the 13 C chemical shifts to the π-electron density in a given molecule. This empirical observation allows to draw a cautious conclusion with respect to the perturbation of the π-electron density upon binding of flavin to a particular apoflavoprotein (see Chapter 3). Reduction of the oxidized flavin by two electrons leads to an upfield shift of the resonances due to C(8), C(6), C(4a) and C(10a). The resonance most affected by reduction is that due to C(4a) (upfield shift of ~35 ppm). From the two electrons added to oxidized flavin 80% is accomodated in the pteridine subring whereas the residual 20% is distributed over the benzene moiety of the flavin molecule. The results further support the view that flavin is a bent molecule in the reduced state.Selectively enriched 13 C flavins and prosthetic groups, free and bound to apoflavoproteins, were also studied (Chapter 2). It is shown that the chemical shifts due to C(2), C(6), C(8) and C(10a) in the oxidized free molecule are strongly influenced by the polarity of the solvent. This observation is ascribed to polarization of the molecule in aqueous solutions. This interpretation is supported by the observed direct (one-bond) 13 C coupling constants measured in polar and apolar solvents. Similarly it could be shown that the conformation of reduced flavin depends also on the polarity of the solvent, i.e. the molecule is less bent in polar than in an apolar solvent. Comparison of the 13 C chemical shifts of free with those of flavin bound to M.elsdenii and A.vinelandii apoflavodoxins revealed that in the oxidized form of the two flavoproteins the flavin forms a hydrogen bond with an amino acid residue of the apoprotein. The interaction with the apoprotein occurs via the 0(2α) atom of flavin. This interpretation is in accord with the observed chemical shift due to C(10a). In contrast to the 0(2α) atom, the 0(4α) atom in M.elsdenii flavodoxin shows a weak interaction with the apoprotein whereas the same atom in A.vinelandii flavodoxin seems to be placed in a rather hydrophobic environment. In the reduced state both flavodoxins possess a more planar structure than free flavin in aqueous solution. In addition these flavodoxins are in the anionic state in the pH range 5.5 to 8.5. Free flavin, on the other hand, shows an ionization constant of 6.7. The facts that the protein-bound flavin is negatively charged, possesses an almost planar structure stabilized by specific interactions with the apoprotein and the relatively apolar environment of the active center indicate that these elements form the basis for the strong reductive power of flavodoxins.In chapter 4 a 1 H NMR study on M.elsdenii flavodoxin is described. The 1 H NMR spectrum shows some well resolved resonances at high field (upfield from the internal standard). These resonances are due to methyl groups being under the influence of ring current effects. Some of these resonance lines could be assigned. It follows from oxidation-reduction experiments that the resonances at -0.72, -0.28 and -0.07 ppm are due to methyl groups located in the neighbourhood of the prosthetic group. The resonance line at -0.07 ppm is assigned to alanine-57 and the lines at - 0.72 and -0.28 ppm to the S-methyl groups of leucine-62. Since the latter two methyl groups are magnetically non-equivalent it must be concluded that the isopropyl moiety of leucine-62 is not free to rotate around the CH-CH 2 bond of leucine.The resonance lines of the three methyl groups are broadened in the radical state supporting the idea that they are located in the active center of the protein. In the two- electron reduced state the resonances due to the methyl groups are slightly downfield shifted indicating attenuation of the ring current effect, probably caused by a slight bending of the isoalloxazine moiety of the prosthetic group. In addition the resonances due to the C(7) and C(8) methyl groups of the prosthetic group could be assigned in the spectrum. These resonances differ only by about 0.5 ppm from the position of those of free flavin. Tryptophan-91, a constituent of the active site of the protein, could be identified in the 1 H NMR spectrum by a combination of various techniques. From the 1 H NMR study it follows that the protein does not undergo a gross conformational change upon reduction.To aid in the assignment of the resonances due to aromatic amino acid residues in the 1 H NMR spectrum of the protein the photochemically induced dynamic nuclear polarization (CIDNP) technique was applied to small size flavoproteins (chapter 5). It was found that the protein-bound flavin did not yield any CIDNP signals. To generate CIDNP signals external free flavins had to be added to solutions of flavoproteins. An interesting observation is that protein-bound flavin could act as a sensitizer in the CIDNP reaction when external, free aromatic amino acids were added to solutions of flavoproteins. This strongly indicated that the active centers of the flavoproteins tested must be accessible to the free amino acids. Several amino acid residues in various flavoproteins could be identified, although the assignment of the aromatic amino acid residues in the sequence of some flavoproteins could not be made. A CIDNP study on free FAD revealed that this molecule exists as an internal complex at neutral pH. This complex is gradually destroyed in solutions of decreasing pH. This result is in agreement with published fluorescence studies.

Report this publication


Seen <100 times