Carbon-13 and nitrogen-15 nuclear magnetic resonance study on the interacton between riboflavin and riboflavin-binding apoprotein

A new procedure was devised for reversibly removing the flavin from flavocytochrome b 2 . It allowed reconstitution with selectively enriched P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the p-electron distribution.The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15 N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13 C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b 2 as in all other known flavoproteins.With respect to N(10), it is more sp 3 -hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp 3 -hybridized than in free FMN, but N(5) exhibits a higher degree of sp 3 -hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane.In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15 Nchemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

Section: Stability Of Flavin-free and Reconstituted Enzymementioning

confidence: 72%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Flavin–protein interactions in flavocytochrome b₂ as studied by NMR after reconstitution of the enzyme with ¹³C‐ and ¹⁵N‐labelled flavin

Fleischmann

Lederer

Müller

et al. 2000

“…The interaction between aporiboflavin‐binding protein and riboflavin in the oxidized and two‐electron reduced state has been addressed by reconstitution of the protein with 13 C‐ and 15 N‐enriched riboflavin derivatives [147]. These studies revealed that the p K a of the N1 atom of the flavin in the reduced state is unusually high (p K a = 7.45).…”

Section: Ion‐exchange Chromatographymentioning

confidence: 99%

Deflavination and reconstitution of flavoproteins

Hefti¹,

Vervoort²,

Berkel³

2003

119

104

Flavoproteins are ubiquitous redox proteins that are involved in many biological processes. In the majority of flavoproteins, the flavin cofactor is tightly but noncovalently bound. Reversible dissociation of flavoproteins into apoprotein and flavin prosthetic group yields valuable insights in flavoprotein folding, function and mechanism. Replacement of the natural cofactor with artificial flavins has proved to be especially useful for the determination of the solvent accessibility, polarity, reaction stereochemistry and dynamic behaviour of flavoprotein active sites. In this review we summarize the advances made in the field of flavoprotein deflavination and reconstitution. Several sophisticated chromatographic procedures to either deflavinate or reconstitute the flavoprotein on a large scale are discussed. In a subset of flavoproteins, the flavin cofactor is covalently attached to the polypeptide chain. Studies from riboflavindeficient expression systems and site-directed mutagenesis suggest that the flavinylation reaction is a post-translational, rather than a cotranslational, process. These genetic approaches have also provided insight into the mechanism of covalent flavinylation and the rationale for this atypical protein modification.

“…Therefore, it is concluded that no hydrogen bond exists between the N(1)H group of protein‐bound FADH 2 and the apoprotein. With respect to the protonated N(1) atom, the reduced enzymes studied in this paper represent so far exceptional cases in published NMR data on flavoproteins [17], where it was found that the N(1) atom in all flavoproteins studied is ionized, with the exception of riboflavin binding protein [42].…”

Section: C‐ and 15n‐nmr Chemical Shifts Of The Isoalloxazine Ringmentioning

confidence: 99%

¹³C‐, ¹⁵N‐ and ³¹P‐NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins

Eisenreich¹,

Kemter²,

Bacher³

et al. 2004

Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U- P-NMR spectroscopy. The enzymes studied showed very similar 31 P-NMR spectra in the oxidized state, consisting of two peaks at )9.8 and )11.5 p.p.m. In the reduced state, the two peaks merge into one apparent peak (at )9.8 p.p.m.). The data are compared with published 31 P-NMR data of enzymes closely related to TrxR.