Molecular relaxation spectroscopy of flavin adenine dinucleotide in wild type and mutant lipoamide dehydrogenase from Azotobacter vinelandii.
The temperature dependence of the fluorescence emission spectra of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii shows that the protein matrix in the vicinity of the prosthetic group is rigid on a nanosecond time scale in a medium of high viscosity (80% glycerol). The active site of a deletion mutant of this enzyme, which lacks 14 C-terminal amino acids, is converted from a solid-state environment (on the nanosecond time scale of fluorescence) into a state where efficient dipolar relaxation takes place at temperatures between 203 and 303 K. In aqueous solution, fast dipolar fluctuations are observed in both proteins. It is shown from fluorescence quenching of the flavin by iodide ions that the prosthetic groups of the mutant protein are partially iodide accessible in contrast to the wild type enzyme. A detailed analysis of the temperature dependence of spectral energies according to continuous relaxation models reveals two distinct relaxation processes in the deletion mutant, which were assigned to solvent and protein dipoles, respectively. From the long-wavelength shifts of the emission spectra upon red-edge excitation, it is demonstrated that the active site of the wild type enzyme has high structural homogeneity in comparison to the deletion mutant. In combination with results obtained by X-ray diffraction studies on crystals of the wild type enzyme, it can be concluded that the C-terminal polypeptide of the A. vinelandii enzyme interacts with the dehydrolipoamide binding site, thereby shielding the flavins from the solvent.
Bastiaens
P I
PI
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
van Hoek
A
A
van Berkel
W J
WJ
de Kok
A
A
Visser
A J
AJ
D016948:Azotobacter vinelandii / Q000201:enzymology*; D001665:Binding Sites; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005454:Fluorescence Polarization; D009154:Mutation; D013057:Spectrum Analysis; D013696:Temperature
D005182:Flavin-Adenine Dinucleotide; D008058:Dihydrolipoamide Dehydrogenase