Detection of Scalar Couplings Across NH···OP and OH···OP Hydrogen Bonds in a Flavoprotein

Hydrogen bonding plays a major role in the tight binding of the FMN cofactor in flavodoxins. The present NMR investigation provides direct experimental evidence for hydrogen bonds involving the phosphate moiety of FMN in Desulfovibrio vulgaris flavodoxin. Several trans-hydrogen bond J couplings between the phosphorus nucleus and backbone amide as well as side chain hydroxyl protons of the apoprotein have been detected. It is shown that relaxation interference between ¹H chemical shift anisotropy and ¹H−³¹P dipolar interactions can also lead to correlations of these nuclei in HMBC spectra. The size of the ^2hJ_PH coupling constants was determined using a simple ³¹P-detected quantitative J correlation experiment. For at least one amide group a scalar three-bond coupling between the phosphorus and nitrogen has been observed in a [¹⁵N,¹H]-TROSY-type ¹⁵N−{³¹P} spin−echo difference experiment. With approximately 1.7 Hz its magnitude is larger than that of the ³¹P−¹H couplings, which ranged from 0.5 to 1.6 Hz.