Electron Microscopy of Cytochrome c Oxidase Crystals: Labeling of Subunit III with a Monomaleimide Undecagold Cluster Compound

Abstract

Two-dimensional crystals of beef heart mitochondrial cytochrome c oxidase dimers were labeled at Cys-115 of subunit III with a monomaleimide derivative of an undecagold cluster compound. The binding site of the gold cluster compound and hence the site of subunit III were identified by image processing of cryoelectron micrographs of the crystals preserved in a mixture of glucose and uranyl acetate. The shape of the cytochrome oxidase dimer can be approximated as a parallelogram which is 44 by 82 A with an included angle of 80 degrees oriented with its long dimension along the a axis of the crystal. Labeling of subunit III was confirmed by a shift in the mobility of approximately 50% of subunit III molecules upon electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Averaged images of undecagold cluster labeled crystals and of unlabeled crystals were calculated; each image represents an average of approximately 17,000 molecules of either labeled or unlabeled cytochrome oxidase. On the basis of a statistical analysis of the differences between the two images, the gold cluster binds along a line 30 degrees from the a axis and 29 A from the center of the dimer. This result is interpreted in the context of other structural studies including the site of cytochrome c binding which Frey and Murray found to be near the a axis and 18 A from the center of the dimer [Frey, T. G., & Murray, J. M. (1994) J. Mol. Biol. 237, 275-297].(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: Implications for morphogenesis and organization of encapsidated RNA

“…1a) with an 11-gold atom cluster that has been used successfully in several previous studies (e.g., refs. [22][23][24]. To obtain specific labeling, we used the maleimidyl derivative, which reacts with cysteines.…”

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6Aresolution

“…Our future experiments will aim at the identification of this substrate‐binding site by using gold‐labelled ubiquinol derivatives. The combination of electron crystallography and gold‐labelled substrates or polypeptides has been used already in studies on the localization of the cytochrome c binding site of mitochondrial oxidase (Frey and Murray, 1994) and the location of subunit III in the same complex (Crum et al ., 1994). Furthermore, our structural analysis will be extended to the recording of tilt series in order to obtain three‐dimensional data of the Cyt bo complex.…”

Monofunctionalized Gold Nanoparticles: Fabrication and Applications

“…Alternatively, monofunctionalized Au 11 clusters were obtained by stoichiometric ligand place-exchange with suitably functionalized phosphine ligands [72] and subsequent chromatography. [77] These facilitated synthetic approaches allowed reliable labeling of a multitude of biomolecules for TEM detection such as proteins, [13,78] antibodies [79] and RNA, [80][81][82] whereby, in the latter case, it was shown that the undecagold label did not interfere with the translation process in the ribosome due to its minuscule size [83,84] -in contrast to its Au 55 counterpart. [85] Recently, N-heterocyclic carbine-monofunctionalized undecagold was reported to have unprecedented thermal stability and catalytic activity in the electroreduction of CO 2 .…”