Conformational dynamics and intersubunit energy transfer in wild-type and mutant lipoamide dehydrogenase from Azotobacter vinelandii. A multidimensional time-resolved polarized fluorescence study.
Time-resolved fluorescence and fluorescence anisotropy data surfaces of flavin adenine dinucleotide bound to lipoamide dehydrogenase from Azotobacter vinelandii in 80% glycerol have been obtained by variation of excitation energy and temperature between 203 and 303 K. The fluorescence kinetics of a deletion mutant lacking 14 COOH-terminal amino acids were compared with the wild-type enzyme to study a possible interaction of the COOH-terminal tail with the active site of the enzyme. The flavin adenine dinucleotide fluorescence in both proteins exhibits a bimodal lifetime distribution as recovered by the maximum entropy method of data analysis. The difference in standard enthalpy and entropy of associated conformational substates was retrieved from the fractional contributions of the two lifetime classes. Activation energies of thermal quenching were obtained that confirm that the isoalloxazines in the deletion mutant are solvent accessible in contrast to the wild-type enzyme. Red-edge spectroscopy in conjunction with variation of temperature provides the necessary experimental axes to interpret the fluorescence depolarization in terms of intersubunit energy transfer rather than reorientational dynamics of the flavins. The results can be explained by a compartmental model that describes the anisotropy decay of a binary, inhomogeneously broadened, homoenergy transfer system. By using this model in a global analysis of the fluorescence anisotropy decay surface, the distance between and relative orientation of the two isoalloxazine rings are elucidated. For the wild-type enzyme, this geometrical information is in agreement with crystallographic data of the A. vinelandii enzyme, whereas the mutual orientation of the subunits in the deletion mutant is slightly altered. In addition, the ambiguity in the direction of the emission transition moment in the isoalloxazine ring is solved. The anisotropy decay parameters also provide information on electronic and dipolar relaxational properties of the flavin active site. The local environment of the prosthetic groups in the deletion mutant of the A. vinelandii enzyme is highly inhomogeneous, and a transition from slow to rapid dipolar relaxation is observed over the measured temperature range. In the highly homogeneous active site of the wild-type enzyme, dipolar relaxation is slowed down beyond the time scale of fluorescence emission at any temperature studied. Our results are in favor of a COOH-terminal polypeptide interacting with the active site, thereby shielding the isoalloxazines from the solvent. This biological system forms a very appropriate tool to test the validity of photophysical models describing homoenergy transfer.
Bastiaens
P I
PI
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
van Hoek
A
A
Benen
J A
JA
Brochon
J C
JC
Visser
A J
AJ
D016948:Azotobacter vinelandii / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D005454:Fluorescence Polarization / Q000379:methods; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D011994:Recombinant Proteins / Q000737:chemistry; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics
D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008058:Dihydrolipoamide Dehydrogenase