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

A new procedure was devised for reversibly removing the flavin from flavocytochrome b(2). It allowed reconstitution with selectively enriched C-13- and N-15-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of P-31. 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 pi-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 N-15-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 C-13-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 stair 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 N-15- chemical 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. P-31-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.