Fluorescence polarization and energy-transfer studies on the pyruvate dehydrogenase complex of Escherichia coli.
We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Escherichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must apply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization. The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (tau c > 3 micros). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.
D055598:Chemical Phenomena; D002621:Chemistry; D008058:Dihydrolipoamide Dehydrogenase; D004735:Energy Transfer; D004801:Eosine Yellowish-(YS); D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D008301:Maleimides; D010084:Oxidation-Reduction; D011768:Pyruvate Dehydrogenase Complex
D005456:Fluorescent Dyes; D008301:Maleimides; D011768:Pyruvate Dehydrogenase Complex; D005182:Flavin-Adenine Dinucleotide; D008058:Dihydrolipoamide Dehydrogenase; D004801:Eosine Yellowish-(YS)