The regulation of the structure and function of flavin (vitamin B2) on binding to apoflavoproteins and its biological implications

1H, 13C, 15N and 31P NMR was applied to a study of free and protein-bound flavins in order to obtain a better insight into the mechanism by which the function of the flavin coenzyme is "tuned" upon binding to apoflavoproteins. A thorough 13C and 15N investigation of free flavins provided a detailed view on the effects of sterical hindrance and polarization on the structure of oxidized and reduced flavins. Moreover, this study resulted in a semi-empirical basis for the interpretation of NMR data obtained on protein-bound flavins. Remarkable results of the study of free flavins are the fact, that oxidized isoalloxazine is not fully planar in an apolar medium, and that fully reduced isoalloxazine is more planar than believed up till now. Unsubstituted reduced isoalloxazine in water has an almost fully sp 2hybridized N(10) and an N(5) atom which is approximately 70% sp 2hybridized. Upon modification the hybridization of N(5) and N(10) can be modulated rather independently due to steric hindrance. The so-called "butterfly" motion has a low activation barrier, which is probably an important feature for the mentioned tuning mechanism.Flavodoxin from Megasphaeraelsdenii is the protein which is most extensively studied in this thesis. By a 13C relaxation study it was shown that the isoalloxazine ring is strongly immobilized upon binding to apoflavodoxin. Also the phosphate group of FMN is strongly immobilized in the interior of the protein. Moreover, the phosphate group is dianionic in the complex, regardless of the redox state of bound FMN. Based on the NMR data, it was shown that the redox potential for the transition semiquinone/hydroquinone is mainly governed by charge interactions. For the first time a quantification of the redox potential modification by the apoprotein could be established, thereby showing that, contrary to literature reports, the redox potential modulation is not governed by steric effects exerted on the isoalloxazine moiety. Both by 31P NMR and 1H NMR it was shown that M.elsdenii flavodoxin acts as a one-electron transferring protein shuttling between the semiquinone and hydroquinone state, because the oxidized state is ruled out as a biologically relevant redox state in this protein due to the introduction of a high activation barrier between the oxidized and semiquinone state. Moreover, the electron transfer mechanism is of an outer sphere type. Based on the NMR data some arguments are presented that a specific complex formation between M.elsdenii flavodoxin and the electron donor or acceptor is not needed for an effective electron transfer. An important part of the active center of M.elsdenii flavodoxin was elucidated using time resolved photochemically induced dynamic nuclear polarization (CIDNP) and modern two-dimensional NMR techniques. The use of the paramagnetic semiquinone state in combination with 2D NMR techniques allowed the generation of NMR spectra of only the active center. Complete assignments were given in both the oxidized and the hydroquinone state for Trp-91, Ala-56 and Tyr-89, which are all very close to the isoalloxazine ring. It was shown that the relative position of Trp-91 with respect to the prosthetic group is slightly different in the two redox states. The active center of flavodoxin from M.elsdenii and Clostridium MP appear to be similar. The only small difference between results of the crystallographic study on Clostridium MP flavodoxin and 1H NMR results on M.elsdenii flavodoxin was observed for the relative position of Ala-56 and Tyr-89.A detailed 13C and 15N NMR study was performed on the complex of riboflavin and Riboflavin Binding Protein from the egg yolk and egg white. Subtle information on hydrogen bonding, conformation of the isoalloxazine ring and solvent accessibility were obtained in oxidized and reduced state. As far as the results could be compared with reported binding studies of riboflavin analogues, the results appear to be in excellent agreement. The pyrimidine ring is exposed to solvent, except for 0(4α) in both redox states. N(10) is forced into the molecular plane, which in turn probably causes N(1) to be somewhat out of the plane. The NMR results are consistent with a partial opening of the protein at pH 6 and a more stable conformation at pH 9. Based on the NMR results a possible function for the complex in the embryonic development is suggested.As an example of the class of dehydrogenases, lipoamide dehydrogenase from Azotobacter vinelandii was studied by 13C NMR. In the oxidized state hydrogen bonds exist to 0(2α) and 0(4α), but the polarization of the isoalloxazine ring probably does not extend to the N(10) atom. The 4 electron reduced protein (both the essential disulfide and the flavin are reduced) contains an essentially planar N(10) atom, and again hydrogen bonds to 0(2α) and 0(4α). The 2 electron reduced protein (EH 2 ) appeared to be particularly interesting. It consists of an equal mixture of protein in which the disulfide is reduced and the flavin oxidized, and protein in which the disulfide is oxidized and the flavin reduced. The results allowed a detailed description of the electronic structure of the two-electron reduced protein. From the NMR results it is concluded that the redox potential of both centers is roughly the same. The results show that if the disulfide is reduced one thiol group is present as an anionic thiol group and extremely close to the C(4a) atom of the flavin. The exchange of reduction equivalents between the two redox centers in EH 2 is slow (<5 s -1). In combination with reported kinetic data, it became evident that NAD +accelerates considerably the exchange of reduction equivalents between the disulfide and the flavin. The results show that this transfer must be implemented in the reaction cycle and offer a nice explanation of some "anomalous" kinetic data.As an example of the class of hydroxylases, p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was studied by 1C NMR. Without substrate the isoalloxazine ring is probably exposed to solvent. Upon substrate binding a drastic change of the polarization of C(2) occurs, which is in accord with the published enzyme-substrate complex as revealed by crystallographic methods. It is suggested, that the active center without substrate is rather mobile in order to facilitate the binding of substrate. A conformational change accompanies the binding of substrate. The active center, as present in the oxidized enzyme-substrate complex, is probably already essentially formed upon reduction of the enzyme in the absence of substrate, which explains the drastically decreased binding rate of substrate after reduction. Upon complex formation' with substrate in the reduced state the N(5) is forced into the molecular plane of flavin. Some arguments are presented suggesting that the sp 2hybridization degree of N(5) and N(10) are the main factors which govern the reactivity (or "activation") of oxygen, but not the πelectron density at the C(4a) center of flavin. An electron transfer from the hydroquinone state towards oxygen probably precedes the formation of a C(4a) peroxyflavin by radical pair combination.

Bibliographic Details

Main Author:	Moonen, C.
Other Authors:	Mueller, F.
Format:	Doctoral thesis biblioteca
Language:	English
Published:	Landbouwhogeschool
Subjects:	chemical structure, choline, cum laude, enzymes, myo-inositol, nicotinamide, pantothenic acid, riboflavin, structure activity relationships, vitamin b complex, enzymen, pantoteenzuur, riboflavine, structuur, structuuractiviteitsrelaties, vitamine b complex,
Online Access:	https://research.wur.nl/en/publications/the-regulation-of-the-structure-and-function-of-flavin-vitamin-b2
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