Open AccessNuclear‐magnetic‐resonance investigation of 15 N‐labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin
Author(s) -
FRANKEN HansDieter,
RÜTERJANS Heinz,
MÜLLER Franz
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb07942.x
Subject(s) - flavin group , chemistry , flavodoxin , chemical shift , crystallography , aqueous solution , hydrogen bond , resonance (particle physics) , photochemistry , molecule , stereochemistry , ferredoxin , organic chemistry , enzyme , physics , particle physics
Flavin derivatives, enriched with 15 N (≊95%) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two‐electron reduced state by the 15 N nuclear magnetic resonance technique. The measurements were conducted with aqueous and chloroform solutions of flavin. A comparison of the chemical shifts of the N(1) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen‐bridge formation to these atoms. The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction. A p K a of 6.8 was determined from pH‐dependent 15 N NMR measurements of the two‐electron reduced flavin molecule. In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution. The 15 N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein. Especially the N(1) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein. In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure. These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium M P. Where possible 15 N‐ 1 H, 15 N‐ 15 N and 13 C‐ 15 N coupling constants were determined. Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein‐bound flavin and for the evaluation of the interaction between flavin and apoprotein. Spin‐lattice relaxation measurements show that the relaxation of the 15 N(3)H group of flavin is predominantly determined by dipole‐dipole interaction. The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.