FLUORESCENCE STUDIES OF THE PYRUVATE-DEHYDROGENASE MULTI-ENZYME COMPLEX FROM ESCHERICHIA-COLI

Reduced lipoic acids on the pyruvate dehydrogenase multienzyme complex from Escherichia coli have been preferentially labeled by making use of the differential reactivity of lipoic acids in different environments. The lipoic acids have been labeled with N-ethylmaleimide, N-(3-pyrene)maleimide (MalPy), and N-[4-(dimethylamino)-3, 5-dinitrophenyl]maleimide (DDPM). As the extent of labeling of the enzyme with MalPy increases, the fluorescence quantum yields decrease, the fluorescence polarization increases, and the emission spectrum changes in a manner indicating increased excimer formation. These results suggest that MalPy on different lipoic acids interact strongly and that some lipoic acids are very close to other lipoic acids. Energy-transfer measurements between MalPy (energy donor) and DDPM (energy acceptor) on different lipoic acids indicate that the average distance between donor and acceptor increases from 24 to 33 A as the ratio of donors to acceptors increases. Energy-transfer measurements between MalPy (energy donor) on lipoic acids in different environments and FAD (energy acceptor) at the catalytic site of the lipoamide dehydrogenase enzyme indicate an intermolecular distance varying from 23 to >47 Å, depending on the particular lipoic acids labeled. Furthermore, the MalPy-labeled lipoic acids appear to move away from the FAD and aggregate with each other as the extent of labeling of the enzyme with MalPy increases. Energy-transfer measurements between thiochrome diphosphate (energy donor) located at the catalytic site of the pyruvate decarboxylase enzyme and DDPM (energy acceptor) labeled lipoic acids indicate little energy transfer with a variety of labeled derivatives; the intermolecular distances calculated range from 38 to >45 Å. These results are consistent with a mechanism involving multiple mobile lipoic acids which transfer acetyl groups and electrons between the three catalytic sites and adjacent lipoic acids. © 1979, American Chemical Society. All rights reserved.