The structure of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. Crystals containing an octameric arrangement of iron-sulphur protein fragments.
We have investigated the structure of two-dimensional crystals from preparations of NADH:ubiquinone oxidoreductase from beef-heart mitochondria. The crystal structure of these crystals was previously determined to be equivalent with two native enzyme molecules per unit cell, i.e. a p2 symmetry [Boekema, E. J., Van Heel, M. G. & Van Bruggen, E. F. J. (1984) Biochim. Biophys. Acta 787, 19-26]. However, the optical diffraction patterns of the crystals displayed a clear fourfold symmetry. A Fourier analysis carried out on the calculated diffraction pattern proved unambiguously that the crystal symmetry was p42(1)2. Following crystallographic rules the unit cell therefore contained eight identical molecules. As a consequence, only a subcomplex of the enzyme rather than the intact enzyme formed the crystal. Electron microscopy of isolated, single molecules of the iron-sulphur protein, a dissociation product of complex I, revealed the presence of square complexes with sides of approximately 15 nm. Since these complexes were indistinguishable from the building blocks (unit cells) of the two-dimensional crystals, the crystals could be composed of Fe-S protein fragments only. The nature of the fragments in the unit cell was probed by immuno-labelling with monovalent antibodies (Fab's), raised against the 75-kDa subunit from the Fe-S protein, followed by image analysis. We found at least four binding sites for the anti-(75-kDa subunit) Fab per unit cell, indicating the presence of at least four copies of the antigen. In order to account for these observations we postulate the hypothesis that the two-dimensional crystals obtained from complex I are composed of iron-sulphur protein molecules in an octameric arrangement.
D000818:Animals; D000936:Antigen-Antibody Complex; D005583:Fourier Analysis; D007140:Immunoglobulin Fab Fragments; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification; D046911:Macromolecular Substances; D008667:Metalloproteins / Q000302:isolation & purification; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology*; D008958:Models, Molecular; D016660:NAD(P)H Dehydrogenase (Quinone); D011487:Protein Conformation; D011808:Quinone Reductases / Q000302:isolation & purification
D000936:Antigen-Antibody Complex; D007140:Immunoglobulin Fab Fragments; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D008667:Metalloproteins; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases