Comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time-resolved polarized flavin fluorescence.
Time-resolved polarized fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter vinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-313 K. Interconversion between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.
Bastiaens
P I
PI
Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
van Hoek
A
A
Wolkers
W F
WF
Brochon
J C
JC
Visser
A J
AJ
D016948:Azotobacter vinelandii / Q000201:enzymology; D008058:Dihydrolipoamide Dehydrogenase / Q000737:chemistry*; D004912:Erythrocytes / Q000201:enzymology; D005415:Flavins / Q000737:chemistry*; D005454:Fluorescence Polarization; D005980:Glutathione Reductase / Q000097:blood / Q000737:chemistry*; D006801:Humans; D011487:Protein Conformation; D013696:Temperature
D005415:Flavins; D008058:Dihydrolipoamide Dehydrogenase; D005980:Glutathione Reductase