Fluorescence energy-transfer studies on the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii.
Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart (greater than or equal to 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis. Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.
D000111:Acetylcysteine / Q000378:metabolism; D001395:Azotobacter / Q000201:enzymology*; D001665:Binding Sites; D004735:Energy Transfer; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005453:Fluorescence; D006898:Hydroxylamines / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013831:Thiamine / Q000031:analogs & derivatives; D008063:Thioctic Acid / Q000031:analogs & derivatives / Q000378:metabolism
D006898:Hydroxylamines; D008301:Maleimides; D009943:Organophosphorus Compounds; D011743:Pyrimidines; D011768:Pyruvate Dehydrogenase Complex; C009460:thiochrome diphosphate; D005182:Flavin-Adenine Dinucleotide; D008063:Thioctic Acid; D000111:Acetylcysteine; D013831:Thiamine