Cryo-electron crystallography of two sub-complexes of bovine complex I reveals the relationship between the membrane and peripheral arms.
NADH:ubiquinone oxidoreductase (complex I) is the first and largest enzyme of the mitochondrial respiratory chain. The low-resolution structure of the complex is known from electron microscopy studies. The general shape of the complex is in the form of an L, with one arm in the membrane and the other peripheral. We have purified complex I from beef heart mitochondria and reconstituted the enzyme into lipid bilayers. Under different conditions, several two-dimensional crystal forms were obtained. Crystals belonging to space groups p222(1) and c12 (unit cell 488 Ax79 A) were obtained at 22 degrees C and contained only the membrane fragment of complex I similar to hydrophobic subcomplex Ibeta but lacking the ND5 subunit. A crystal form with larger unit cell (534 Ax81 A, space group c12) produced at 4 degrees C contained both the peripheral and membrane arms of the enzyme, except that ND5 was missing. Projection maps from frozen hydrated samples were calculated for all crystal forms. By comparing two different c12 crystal forms, extra electron density in the projection map of large crystal form was assigned to the peripheral arm of the enzyme. One of the features of the map is a deep, channel-like, cleft next to peripheral arm. Comparison with available structures of the intact enzyme indicates that large hydrophobic subunit ND5 is situated at the distal end of the membrane domain. Possible locations of subunit ND4 and of other subunits in the membrane domain are proposed. Implications of our findings for the mechanism of proton pumping by complex I are discussed.
Sazanov
L A
LA
Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 2XY, UK.
Walker
J E
JE
D000818:Animals; D002417:Cattle; D020285:Cryoelectron Microscopy; D003460:Crystallization; D003902:Detergents / Q000737:chemistry / Q000378:metabolism; D042967:Electron Transport Complex I; D004591:Electrophoresis, Polyacrylamide Gel; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D008823:Micelles; D008931:Mitochondria, Muscle / Q000201:enzymology; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D020836:Protein Structure, Quaternary; D013696:Temperature
D003902:Detergents; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D010446:Peptide Fragments; D009247:NADH, NADPH Oxidoreductases; D042967:Electron Transport Complex I