The molecular morphology of bovine heart mitochondrial NADH-ubiquinone reductase. Cross-linking with dithiobis(succinimidyl propionate).
The molecular morphology of NADH-ubiquinone reductase (complex I) was investigated by cross-linking with the cleavable bifunctional reagent, dithiobis(succinimidyl propionate). Cross-linking inhibits the following activities of the complex--NADH----3-acetylpyridine adenine dinucleotide (oxidized), NADH----2,6-dichloroindophenol, NADH----ferricyanide, and NADH----menadione--to different degrees with the greatest inhibition occurring with either ferricyanide or 3-acetylpyridine adenine dinucleotide as electron acceptor. Addition of 150 microM NADH affords partial protection from inhibition. Cross-linking quenches the FMN fluorescence of complex I (288 nm excitation/515 nm emission), and addition of 150 microM NADH greatly reduces the quenching. Treatment of complex I (1 mg/ml) for 2 min with dithiobis(succinimidyl propionate) (0.2 mg/ml) at 4 degrees C revealed a cross-linked product consisting of the following seven subunits: 75-80, 53-57, 42, 33-35, 24-27, 17-18, and 12.5-15.5 kDa. Five minutes of treatment cross-linked the unidentified polypeptides of 69 and 51 kDa to six of the seven complex I subunits, but the 12.5-15.5-kDa subunit may be missing from this cross-linked product, while 15 min of treatment cross-linked additional unidentified polypeptides of 177, 107, 72, and 63 kDa. Since longer times of cross-linking result in a larger number of unidentifiable polypeptide spots, the shorter cross-linking time results are taken as a more accurate picture of the native enzyme conformation. This would indicate that within complex I the following subunits are within 12 A of each other at one or more points in space: 75-80, 53-57, 42-45, 33-35, 24-27, 17-18, and, perhaps, 12.5-15.5 kDa. These subunits represent portions of all three fractions of the enzyme, i.e. flavoprotein, iron-protein, and insoluble or hydrophobic fractions.
D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008929:Mitochondria, Heart / Q000201:enzymology*; D016660:NAD(P)H Dehydrogenase (Quinone); D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation; D011808:Quinone Reductases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013388:Succinimides
D003432:Cross-Linking Reagents; D013388:Succinimides; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases; C011240:dithiobis(succinimidylpropionate)