Open AccessProtein Mobility Inside Pyruvate Dehydrogenase Complexes as Reflected by Laser‐Pulse Fluorometry
Author(s) -
GRANDE Hans J.,
VISSER Antonie J. W. G.,
VEEGER Cees
Publication year - 1980
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1980.tb04582.x
Subject(s) - chemistry , flavin group , azotobacter vinelandii , pyruvate dehydrogenase complex , flavin adenine dinucleotide , fluorophore , dehydrogenase , cofactor , fluorescence , photochemistry , analytical chemistry (journal) , crystallography , enzyme , biochemistry , physics , chromatography , nitrogen , nitrogenase , organic chemistry , nitrogen fixation , quantum mechanics
The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two‐exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode‐locked argon‐ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three‐component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme‐free complex is compared with complex to which Mg 2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11°C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipoamide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi‐enzyme complex is proposed.