Electron microscopy of cytochrome c oxidase crystals: labeling of subunit III with a monomaleimide undecagold cluster compound.
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)
Crum
J
J
Department of Biology, San Diego State University, California 92182-4614.
Gruys
K J
KJ
Frey
T G
TG
D000818:Animals; D002417:Cattle; D003461:Crystallography; D003576:Electron Transport Complex IV / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D006046:Gold; D007091:Image Processing, Computer-Assisted; D008301:Maleimides; D008929:Mitochondria, Heart / Q000201:enzymology; D050607:Organogold Compounds; D009942:Organometallic Compounds
D008301:Maleimides; D050607:Organogold Compounds; D009942:Organometallic Compounds; C077615:monomaleimido gold; C035797:undecagold; D006046:Gold; D003576:Electron Transport Complex IV