31P-NMR spectroscopy of human and Paracoccus denitrificans electron transfer flavoproteins, and 13C- and 15N-NMR spectroscopy of human electron transfer flavoprotein in the oxidised and reduced states

Human and Paracoccus denitrificans wild-type electron transfer flavoproteins have been investigated by P-31-NMR in the oxidised and reduced states. The P-31 chemical shifts of the diphosphate moiety of the protein-bound FAD were similar in the proteins and were independent of the redox state. The chemical shifts were remarkably similar to those of ferredoxin-NADP(+) reductase and, to a lesser degree, with those of NADPH-cytochrome P-450 reductase. The wild-type human electron transfer apoprotein was reconstituted with [2,4a-C-13(2)]FAD, [4,10a-C-13(2)]FAD, or [U-N-15(4)]FAD. The reconstituted proteins were studied by C-13- and N-15-NMR techniques in the oxidised and reduced states. The chemical shifts were compared with those of free flavin in aqueous solution or in chloroform, and those of flavoproteins published in the literature. In the oxidised state, strong hydrogen bonds exist between residues of the apoprotein and C(2)O and N(5) of FAD. The N(1) atom is also hydrogen bonded and, as shown by X-ray data, involves the C'(4)OH group of FAD. The sp(2) hybridisation of N(10) is small compared to other flavoproteins. In the reduced state, there are strong hydrogen bonds involving C(2)O and N(5) of FAD. The N(1) atom is ionised as observed also in other flavoproteins when investigated by NMR. The intramolecular hydrogen bond between the C'(4)-OH group and the N(1) atom of FAD is maintained in the reduced state, suggesting an involvement in the stabilisation of a certain configuration of the diphosphate group of protein-bound FAD in both redox states. The N(10) atom in the reduced protein is highly sp(3) hybridised in comparison to those of other flavoproteins.