Structures From Xray Diffraction

5.1. Cytochrome c Oxidase

When the first atomic structure of a eukaryotic cytochrome oxidase determined by X-ray crystallography was published in 1995 (74), its structure had previously been determined in 2D projection at approximately 8 to 10 Â resolution (79) and in 3 dimensions at approximately 15 Â resolution (35). This is too low a resolution to discern subunit boundaries, let alone trace the polypeptide chains, but a number of structural features had been deduced by specimen preparation to contrast different domains selectively, by various labeling experiments, and by comparing the structures of both 2D crystal forms, the monomer form, and the dimer form. The transmembrane a-helices predicted by hydropathy plots based on amino acid sequences proved to be fairly accurate, and according to the X-ray model, they separate the molecule into two hydrophilic domains that protrude 35 to 40 Â beyond the lipid bilayer into the intermembrane space and into the matrix space of mitochondria. This is in contrast to the marked asymmetric distribution of protein mass, 60 Â into the intermembrane space and less than 10 Â into the matrix space, proposed from 3D reconstructions by electron crystallography (18,42,79). It is difficult to reconcile these and accept the X-ray model as being more accurate. On should note, however, that Frey et al. correctly determined that the matrix side domain projected 20 to 30 Â based upon the lengths of shadows cast in specimens that had been freeze-dried and shadowed with platinum-carbon (32-34). This highlights the importance of using different specimen preparation techniques in studying complex biological structures by electron microscopy.

Although in projection, the dimers observed in 2D crystals by cryoelectron microscopy appear very similar to those generated from the X-ray coordinants (obtained from the Protein Data Bank and displayed with the program RasMol; see Figure 7) (75), comparison of their sizes indicates that they must be different structures. The maximum dimension parallel to the membrane of dimers in the 2D crystals is approximately 100 A, the length of the a crystal axis along which the molecules are aligned. The X-ray model, on the other hand, has a maximum dimension of approximately 150 A. Thus, it appears that the dimer in 2D crystals must be a more compact structure with the individual monomers more closely aligned than the crystallographic dimers in the 3D crystals used to determine the structure by X-ray diffraction. This is also indicated by the fact that the dimer in 2D crystals has its highest concentration of mass around the 2-fold axis, while the dimer in the 3D crystals is less densely packed in this region. The size and shape of a cytochrome oxidase monomer observed in 2D crystals compares more favorably with the X-ray structure (Figure 5). At 95 x 53 A, the structure determined by electron microscopy (35) is somewhat smaller than the 110 x 63 A structure measured from X-ray coordi-nants, but this difference can be explained by the ambiguity in determining the molecular boundary in a 2D projection.

The sites identified by electron microscopy following specific labeling with Fabs, cytochrome c, and a monomaleimide undecagold cluster are, for the most part, confirmed by the atomic structure determined by X-ray crystallography. As shown in Figure 5, the cytochrome c binding site in images of cytochrome oxidase monomers is in essentially the same position as the binding site in the X-ray structure deduced from the positions of Cys-115 of subunit III and the acidic residues of subunit II that have been shown to bind to opposite surfaces of cytochrome c in the active site (9,25). Although the position of Cys-115 of subunit III in the X-ray structure appears to be quite distant from the peak identified for the undecagold cluster compound bound to Cys-115 in the low resolution structure determined by electron crystallography (16), one must remember that the dimer observed in 2D crystals by electron microscopy is much more compact than the dimer found in 3D crystals by X-ray diffraction. In order to compare these 2 structures, the monomers in the X-ray structure must each be moved approximately 25 Â towards one another placing Cys-115 of subunit III within the 15 Â length of the link between the undecagold cluster observed by electron microscopy and the maleimide group bound to the sulfhydryl of Cys-115.

5.2. Cytochrome c Reductase

The low resolution structure of the Neurospora cytochrome c reductase (cyto-chrome bc1 complex) determined by electron microscopy is very similar the atomic structure of the beef heart mitochondrial enzyme determined by X-ray diffraction (49,85,88). As shown in Figure 6, the structure calculated from electron micrographs of 2D crystals displays an asymmetric distribution of mass protruding 30 A on one side of the bilayer and 70 A on the other with a 50 A domain within the lipid bilayer (56,81). The smaller domain protruding 30 A was identified as the

hydrophilic subunits of cytochrome q and the Rieske iron sulfur protein (subunits IV and V in Figure 7), and the larger domain as the core subunits I and II based upon the structure of a subcomplex lacking sub-units I and II (47,52). These assignments are confirmed in the X-ray structure in which the cytochrome c1 and Rieske iron sulfur domains extend 30 A beyond the bilayer and the core subunits 70 A beyond. The dimensions of the cytochrome c reductase dimer are also similar: 120 x 75 A in the electron microscopy structure versus 143 x 102 A in the X-ray structure. Here, the somewhat smaller structure determined by electron microscopy can be attributed to: (i) shrinkage when the 2D crystals are prepared for electron microscopy by negative staining; and/or (ii) the fact that the structure determined by electron microscopy is of the Neurospora enzyme and that determined by X-ray diffraction is of the beef heart enzyme. The structure of cytochrome b6fin projection (8) is very similar to the structure of the cytochrome bc1 subcomplex calculated from atomic coordinants, but a 3D structure of the cytochrome b6fcomplex is not yet available.

examples of heme proteins described here have yielded only low resolution structures, however, and the complete structures were obtained by X-ray crystallography of 3D crystals. The failure of electron crystallography to produce high resolution structures of these enzymes may be explained in part by their large sizes. Cytochrome c oxidase has a molecular weight of 200 000 (400 000 for the dimer form), and cyto-chrome c reductase has a molecular weight of 250 000 (500 000 for the dimer), and both contain many different polypeptide subunits. Nevertheless, low resolution models calculated from electron micrographs provided early insight into the structures of these critically important enzymes. Identification of functional sites and domains by specific labeling and by crystallization of subcomplexes also proved valuable in elucidating the structures of these enzymes and in explaining some aspects of their function. Furthermore, the technique of electron crystallography has been proven to be capable of determining the atomic structures of a number of proteins and will surely prove useful in elucidating the structures of other heme-con-taining membrane proteins.

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