The interactions between apoflavoproteins and their coenzymes as studied by nuclear magnetic resonance techniques

Research output: Thesisinternal PhD, WU


High resolution <sup><font size="-1">13</font></SUP>C, <sup><font size="-1">15</font></SUP>N, <sup><font size="-1">17</font></SUP>O and <sup><font size="-1">31</font></SUP>P NMR techniques have been applied to study the structure of free and protein-bound flavins. These techniques yield information on the molecular and sub-molecular level and hence on the mechanism by which the flavin coenzyme is tuned to its specific function. A large part of the thesis deals with the interaction between FMN and apoflavodoxins from several sources <u>i.e.</u><u>Megasphaera elsdenii</u> , <u>Clostridium MP</u> , <u>Azotobacter vinelandii</u> and <u>Desulfovibrio vulgaris</u><sup><font size="-1">13</font></SUP>It is shown in these studies that subtle differences in the interaction between apoflavodoxin and FMN reveal themselves by different <sup><font size="-1">13</font></SUP>C, <sup><font size="-1">15</font></SUP>N or <sup><font size="-1">31</font></SUP>P chemical shifts.<p>It is shown in chapter 2 that on binding of FMN to the apoflavodoxin from <u>Desulfovibrio vulgaris</u> the isoalloxazine ring of FMN becomes strongly polarized in the oxidized state. Electron density is reallocated from the benzene ring through mesomeric structures towards the C(2) carbonyl group. The pyrimidine part of the molecule is buried in the protein and is not accessible for bulk solvent. On two-electron reduction the isoalloxazine ring maintains its coplanar structure. The isoalloxazine ring is ionized, <u>i.e.</u> carrying a negative charge at N(1), in the pH region between 6.0 and 8.5. Two-electron reduction of the protein leads to a large πelectron density increase in the benzene subnucleus of bound FMN compared to free FMN. This increase in πelectron density is most likely due to the electron-donating effect of the N(5) and N(10) atoms.<p>In chapter 3 the interaction between FMN and the apoprotein of three other flavodoxins is studied, <u>i.e.</u> from <u>Azotobacter vinelandii</u> , <u>Megasphaera elsdenii</u> and <u>Clostridium MP</u> . The results show the virtually identical interaction between the prosthetic group and the apoflavodoxins <u>from M.elsdenii</u> and <u>C.MP</u> . Subtle but significant differences are found between these two flavodoxins and the flavodoxins from <u>A.vinelandii</u> and <u>D.vulgaris</u> , <u>e.g.</u> in <u>A.vinelandii</u> flavodoxin the <sup><font size="-1">15</font></SUP>N chemical shift indicates that the N(10) atom is out of plane in both redox states. This in contrast to the other three flavodoxins. In all four flavodoxins the pteridine part of the molecule is shielded from solvent in both redox states. The N(5) atom, forms a weak hydrogen bond in the oxidized state in <u>A.vinelandii</u> and <u>D.vulgaris</u> flavodoxin, a rather surprising result. No hydrogen bond towards N(5) is found in <u>C.MP</u> and in <u>M.elsdenii</u> flavodoxin. The <sup><font size="-1">13</font></SUP>C and <sup><font size="-1">15</font></SUP>N results suggest that in <u>A.vinelandii</u> flavodoxin the prosthetic group is bound in a more hydrophobic environment than in the other flavodoxins. In the two-electron reduced state all four flavodoxins are ionized. A strong hydrogen bond towards N(5) is observed in all four flavodoxins. The isoalloxazine ring is coplanar, except in <u>A.vinelandii</u> flavodoxin where the N(10) atom is slightly out of plane. The C(4) carbonyl group does not form a hydrogen bond with the apoprotein and as a result the O(4α) cannot allocate electron density to such an extent as free flavin. Possibly Coulomb repulsive forces from a carbonyl group of a peptide group of the apoprotein, which forms a strong hydrogen bond with the N(5) atom of the isoalloxazine ring, play also an important role. No clear correlation between redox potential and either <sup><font size="-1">13</font></SUP>C or <sup><font size="-1">15</font></SUP>N chemical shifts in the reduced state was found with the four flavodoxins although they differ by about 120 mV in redox potential for the semiquinone- hydroquinone transition.<p>Nuclear Overhauser Effect measurements reveal that the isoalloxazine ring is rigidly bound in both the oxidized and in the twoelectron reduced state.<p>The interaction between a modified flavocoenzyme, i.e. riboflavin 3',5'-bisphosphate, and <u>Megasphaera elsdenii</u> apoflavodoxin was studied In the three redox states (chapter 4). This flavin analog binds rather well to the apoprotein. It was expected that the introduction of an extra phosphate group might influence the redox potentials and this was indeed observed albeit less than expected. This is explained by the observation that the 3'-phosphate is protonated on binding to the apoprotein. The interactions between the isoalloxazine ring and the apoprotein are hardly influenced by the introduction of the extra phosphate group.<p>In chapter 5 it is shown that when FMN is bound to bacterial luciferase the benzene part of the isoalloxazine ring is in a hydrophobic environment. The polarization of the ring system is much weaker than in the flavodoxins. The N(5) atom is strongly hydrogen bonded in the oxidized state. On two-electron reduction the flavin becomes anionic in the physiological pH region. The negative charge at N(1) is possibly counteracted by a positively charged group on the protein. The N(5) atom in luciferase-bound FMHN <sup>-</SUP>is highly sp <sup><font size="-1">2</font></SUP>hybridized rendering an almost planar structure of the prosthetic group. There is one specific strong binding site for FMNH <sup>-</SUP>per dimeric luciferase molecule. Excess reduced flavin can, however, bind to luciferase in a nonspecific manner.<p>In chapter 6 the true <sup><font size="-1">13</font></SUP>C spectrum of intermediate iI of luciferase is identified. In contrast to published results the C(4a) atom of the intermediate resonates at 82.5 ppm, and not at 74 ppm. The intermediate possesses an almost planar structure as deduced by comparison with model studies with only the C(4a) displaced out of plane. The resonance at 74 ppm, previously assigned to the intermediate, is due to a contamination contained in ethylene glycol-d <sub><font size="-1">6</font></sub> .<p>As an example of the class of hydroxylases, para-hydroxybenzoate hydroxylase from <u>Pseudomonas fluorescens</u> was studied by <sup><font size="-1">13</font></SUP>C, <sup><font size="-1">15</font></SUP>N and <sup><font size="-1">31</font></SUP>P NMR (chapter 7). In the substrate-free enzyme the isoalloxazine ring is probably solvent accessible. Upon binding of substrate the isoalloxazine ring becomes shielded from water. The N(1) and C(2) shift strongly upfield, probably as a consequence of the altered position of a helix dipole in the enzyme-substrate complex. In the reduced state the isoalloxazine ring is not solvent accessible in the substrate-free enzyme. The flavin-molecule carries a negative charge at N(1). The isoalloxazine ring is coplanar when bound to the protein. The binding of substrate causes the resonances of the N(1), C(10a) and N(10) to shift strongly upfield due to the interaction with a helix dipole.<p>As an example of the class of dehydrogenases, mercuric reductase from <u>Pseudomonas aeruginosa</u> was investigated by <sup><font size="-1">13</font></SUP>C NMR and <sup><font size="-1">31</font></SUP>P NMR (chapter 8). In the oxidized state the <sup><font size="-1">13</font></SUP>C chemical shifts are not much different from those found in free flavin in water. The N(5) atom in mercuric reductase probably lacks a hydrogen bond in the oxidized state.<p>The protein consists of an oxidized flavin and a reduced disulfide in the two-electron reduced state. The thiolate anion is located in the immediate neighbourhood of the C(4a) atom. The binding of NADPH to the two-electron reduced enzyme leads to an upfield shift of the <sup><font size="-1">13</font></SUP>C resonances, which may indicate charge transfer from the bound NADPH to the isoalloxazine molecule. The binding of NADP <sup><font size="-1">+</font></SUP>to the two-electron reduced enzyme leads to a fast intramolecular electron transfer between (probably) the reduced disulfide and the NADP <sup><font size="-1">+</font></SUP>molecule. In the four-electron reduced state the isoalloxazine ring is ionized and bent at the N(5) position.<p><sup><font size="-1">31</font></SUP>P NMR studies revealed that mercuric reductase has an extra covalently bound, solvent accessible, phosphate group besides the pyrophosphate group of the protein-bound FAD.<p><sup><font size="-1">17</font></SUP>O NMR studies on free flavins show that besides hydrogen bonding also a high dielectric constant is needed to polarize both carbonyl groups (chapter 9). The spectrum of the two-electron reduced [2α- <sup><font size="-1">17</font></SUP>O] TARFH <sub><font size="-1">2</font></sub> shows a doublet, which indicates the existence of isomers in reduced flavin in an apolar solvent.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Mueller, F., Promotor, External person
Award date26 Mar 1986
Place of PublicationWageningen
Publication statusPublished - 1986


  • chlorophyll
  • flavonoids
  • nuclear magnetic resonance
  • nuclear magnetic resonance spectroscopy
  • porphyrins
  • steroids

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