Detection of scalar couplings across NH center dot center dot center dot OP and OH center dot center dot center dot OP hydrogen bonds in a flavoprotein

Hydrogen bonding plays a major role in the tight binding of the FMN cofacto r in flavodoxins. The present NMR investigation provides direct experimenta l evidence for hydrogen bonds involving the phosphate moiety of FMN in Desu lfovibrio vulgaris flavodoxin. Several trans-hydrogen bond J couplings betw een the phosphorus nucleus and backbone amide as well as side chain hydroxy l protons of the apoprotein have been detected. It is shown that relaxation interference between H-1 chemical shift anisotropy and H-1-P-31 dipolar in teractions can also lead to correlations of these nuclei in HMBC spectra. T he size of the (2h)J(PH) coupling constants was determined using a simple P -31-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 [N-15,H-1]-TROSY-type N-15-{P-31}) spin-echo difference experiment. With approximately 1.7 Hz its magnitude is larger than that of the P-31-H-1 couplings, which ranged from 0.5 to 1.6 Hz.