Electron microscopy of cytochrome c oxidase crystals. Monomer-dimer relationship and cytochrome c binding site.
Cytochrome c oxidase was isolated from beef heart mitochondria by detergent extraction yielding two different crystal forms. Extraction with Triton detergents produced vesicular crystals with two-dimensional crystalline arrays of cytochrome c oxidase dimers while extraction with sodium deoxycholate produced crystalline sheets of cytochrome c oxidase monomers. The structures of both crystal forms were determined in two-dimensional projection along an axis normal to the plane of the membrane by cryoelectron microscopy of crystals embedded in vitreous ice (frozen-hydrated). The projection structures of unstained frozen hydrated monomers and of dimers are similar to the structures of the crystals in negative stain. The molecular outline of dimers can be approximated by a parallelogram 44 A by 82 A with an included angle of 80 degrees. Monomers are less regular consisting of two large domains with a smaller domain at one end and a total length of approximately 82 A. Comparison of the two structures reveals the orientation of cytochrome c oxidase monomers within dimers, an orientation which is different from earlier models of monomer-monomer interaction, and suggests a very close interaction between monomers when they associate to form dimers. The crystalline sheets of cytochrome c oxidase monomers bind tightly the small peripheral membrane protein substrate, cytochrome c, and this binding accentuates a tendency of these crystals to stack upon one another. Images of crystals of the cytochrome c oxidase/cytochrome c complex were analyzed by crosscorrelation analysis versus the monomer crystal image. Two types of two-layer crystals have been identified. Both types have one layer rotated by 180 degrees with respect to the other, but they differ in the shifts of origin along crystal axes of the two layers. Difference images formed by subtracting simulated multilayered crystal images (which have no bound cytochrome c) from the complex crystals (cytochrome c oxidase plus cytochrome c) contain one positive difference peak for each cytochrome oxidase monomer within a unit cell. Comparison of the difference peak loci among the different crystal forms is interpreted based upon a consensus cytochrome c binding site in the single layer cytochrome oxidase monomer crystal image.
Frey
T G
TG
Department of Biology, San Diego State University, CA 92182-0057.
Murray
J M
JM
D000818:Animals; D002417:Cattle; D003460:Crystallization; D003461:Crystallography; D003574:Cytochrome c Group / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology; D011487:Protein Conformation
D003574:Cytochrome c Group; D008051:Lipid Bilayers; D008565:Membrane Proteins; D003576:Electron Transport Complex IV