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 th e complex is in the form of an L, with one arm in the membrane and the othe r peripheral. We have purified complex I from beef heart mitochondria and r econstituted the enzyme into lipid bilayers. Under different conditions, se veral two-dimensional crystal forms were obtained. Crystals belonging to sp ace groups p222(1) and c12 (unit cell 488 Angstrom x 79 Angstrom) were obta ined at 22 degrees C and contained only the membrane fragment of complex I similar to hydrophobic subcomplex I beta but lacking the ND5 subunit. A cry stal form with larger unit cell (534 Angstrom x 81 Angstrom, space group c1 2) produced at 4 degrees C contained both the peripheral and membrane arms of the enzyme, except that ND5 was missing. Projection maps from frozen hyd rated samples were calculated for all crystal forms. By comparing two diffe rent c12 crystal forms, extra electron density in the projection map of lar ge crystal form was assigned to the peripheral arm of the enzyme. One of th e features of the map is a deep, channellike, cleft next to peripheral arm. Comparison with available structures of the intact enzyme indicates that l arge hydrophobic subunit ND5 is situated at the distal end of the membrane domain. Possible locations of subunit ND4 and of other subunits in the memb rane domain are proposed. Implications of our findings for the mechanism of proton pumping by complex I are discussed. (C) 2000 Academic Press.