Flavin-protein interactions in flavocytochrome b(2) as studied by NMR after reconstitution of the enzyme with C-13- and N-15-labelled flavin

A new procedure was devised for reversibly removing the flavin from flavocy tochrome b(2). It allowed reconstitution with selectively enriched C-13- an d N-15-labelled FMN for an NMR analysis of the chemical shifts of the enric hed positions as well as that of P-31. From these measurements, it was poss ible 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) f orm hydrogen bonds to the apoenzyme in both redox states. Nevertheless, acc ording to N-15-chemical shifts, the bond from the protein to N(3) is very w eak in both redox states, whereas that to N(5) is strong for the oxidized s tate, and is weakened upon flavin reduction. On the other hand, the C-13-NM R results indicate that the C(2) and C(4) carbonyl oxygens form stronger hy drogen bonds with the enzyme than most other flavoproteins in both redox st ates. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measu red for N(5) in the reduced stair due to lack of resolution, N(5) is clearl y 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 th an in free FMN, whereas the other nitrogen atoms show a nearly planar struc ture. 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)-hy bridization than N(10), which is only slightly shifted out of the isoalloxa zine 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 characteristi c 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 enzym e bound FMN compared to the free flavin in water, indicating a strong inter action of the phosphate group with the apoenzyme.