Cross-linking of the electron-transfer flavoprotein to electron-transfer flavoprotein-ubiquinone oxidoreductase with heterobifunctional reagents.
The mitochondrial electron-transfer flavoprotein (ETF) is a heterodimer containing only one FAD. In previous work on the structure-function relationships of ETF, its interaction with the general acyl-CoA dehydrogenase (GAD) was studied by chemical cross-linking with heterobifunctional reagents [D. J. Steenkamp (1987) Biochem. J. 243, 519-524]. GAD whose lysine residues were substituted with 3-(2-pyridyldithio)propionyl groups was preferentially cross-linked to the small subunit of ETF, the lysine residues of which had been substituted with 4-mercaptobutyramidine (MBA) groups. This work was extended to the interaction of ETF with ETF-ubiquinone oxidoreductase (ETF-Q ox). ETF-Q ox was partially inactivated by modification with N-succinimidyl 3-(2-pyridyldithio)propionate to introduce pyridyl disulphide structures. A similar modification of ETF caused a large increase in the apparent Michaelis constant of ETF-Q ox for modified ETF owing to the loss of positive charge on some critical lysines of ETF. When ETF-Q ox was modified with 2-iminothiolane to introduce 4-mercaptobutyramidine groups, only a minor effect on the activity of the enzyme was observed. To retain the positive charges on the lysine residues of ETF, pyridyl disulphide structures were introduced by treating ETF with 2-iminothiolane in the presence of 2,2'-dithiodipyridyl. The electron-transfer activity of the resultant ETF preparation containing 4-(2-pyridyldithio)butyramidine (PDBA) groups was only slightly affected. When ETF-Q ox substituted with MBA groups was mixed with ETF bearing PDBA groups, at least 70% of the cross-links formed between the two proteins were between the small subunit of ETF and ETF-Q ox. ETF-Q ox, therefore, interacts predominantly with the same subunit of ETF as GAD. Variables which affect the selectivity of ETF-Q ox cross-linking to the subunits of ETF are considered.
Steenkamp
D J
DJ
Department of Chemical Pathology, University of Cape Town Medical School, Observatory, South Africa.
D044944:Acyl-CoA Dehydrogenases / Q000378:metabolism*; D000596:Amino Acids; D003432:Cross-Linking Reagents; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D004586:Electrophoresis; D005420:Flavoproteins / Q000378:metabolism*; D007096:Imidoesters; D007506:Iron-Sulfur Proteins; D008239:Lysine; D009097:Multienzyme Complexes / Q000378:metabolism*; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011725:Pyridines; D013329:Structure-Activity Relationship; D013388:Succinimides; D013438:Sulfhydryl Compounds; D014302:Trinitrobenzenesulfonic Acid
D000596:Amino Acids; D003432:Cross-Linking Reagents; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D007096:Imidoesters; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; D011725:Pyridines; D013388:Succinimides; D013438:Sulfhydryl Compounds; C058185:4-(2-pyridyldithio)butyramidine; C058184:4-mercaptobutyramidine; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C005905:methyl 4-mercaptobutyrimidate; D014302:Trinitrobenzenesulfonic Acid; D044944:Acyl-CoA Dehydrogenases; D000587:Oxidoreductases Acting on CH-NH Group Donors; C023120:electron-transferring-flavoprotein dehydrogenase; D008239:Lysine