Protein Mobility Inside Pyruvate Dehydrogenase Complexes as Reflected by Laser‐Pulse Fluorometry

Abstract

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 Azotohacter vinelundii 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 . virzelundii complexes when coenzyme-free complex is compared with complex to which Mg2 + 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.A number of fluorescence studies on flavinadenine dinucleotide either free in solution or bound to a protein have been reported [I -61. Changes in fluorescence properties were used to detect conformational changes. Energy transfer studies to and from the flavin in lipoamide dehydrogenase and in the pyruvate dehydrogenase complex have been applied to obtain distance information in proteins and protein complexes. Natural donors, like tryptophan [2] as well as covalently bound fluorescent labels [7-101, have been used for this purpose.Previous lifetime studies employing the singlephoton counting technique on lipoamide dehydrogenase from pig heart by Visser et al. [ I l l revealed that the fluorescence decay of the bound flavins was consistent with the presence of two lifetimes. This result was confirmed by phase fluorometer data showing non-identical apparent lifetimes from phase and modulation [12]. Since lipoamide dehydrogenase is an enzyme consisting of two identical peptide chains and since the amplitudes as determined by single-~~ Ahhreviutzon TPP, thiamin diphosphate.photon counting were about identical, it was proposed that the two monomers have bound their respective FAD in a slightly different manner.The presence of two lifetimes is not only interesting, but also complicates considerably the conclusions from, for example, energy transfer. For this reason phase and modulation lifetimes of FAD in lipoamide dehydrogenase free and bound to pyruvate dehydrogenase complexes from other sources were compared [9,12]. The FAD in pyruvate dehydrogenase complex isolated from pig heart appeared to show even bigger differences in phase and modulation lifetimes...

Flavin binding site differences between lipoamide dehydrogenase and glutathione reductase as revealed by static and time‐resolved flavin fluorescence

“…In lipoamide dehydrogenase, however, the fluorescence quantum yield is relatively high. Also the average fluorescence lifetime is quite long [6]. Our timeresolved fluorescence and fluorescence anisotropy results provide clear evidence, that the flavin microenvironments in both enzymes are different.…”

Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli. Genetic reconstruction in vitro and 1H-n.m.r. spectroscopy

“…The position of this sequence in the E2o chain corresponds with that of the much shorter (alanine + proline)-rich sequence (residues 370-377) of the E2p chain referred to above, although the sequences of the E2p and E2o chains are not otherwise markedly homologous in this region (Spencer et al, 1984). If this region of the E2p and E2o polypeptide chains, which links the E3-binding domain to the inner core-forming catalytic domain, does enjoy some degree of conformational freedom in the intact complexes, it might provide a structural basis for interpreting the hitherto puzzling fluorescence data which suggest that E3 bound to the PDH complex is quite mobile (Grande et at., 1980). Further genetic reconstructions of the E2p and E2o polypeptide chains should enable unequivocal answers to these questions to be obtained.…”

On the Mobility of Riboflavin 5′‐Phosphate in Megasphaera elsdenii Flavodoxin as Studied by 13C‐Nuclear‐Magnetic‐Resonance Relaxation

“…elsdenii is tightly associated with the apoprotein. This is an interesting observation with regard to nanosecond fluorescence data on the pyruvate dehydrogenase complex [42] for which it was found that the protein-bound flavin possesses a certain internal freedom on the nanosecond timescale. It is possible that these observations are linked to the specific biological functions of the proteins under consideration.…”