Selective labeling of beef heart cytochrome oxidase subunit III with eosin‐5‐maleimide

Müller, Michèle; Azzi, Angelo

doi:10.1016/0014-5793(85)80664-5

Cited by 13 publications

(3 citation statements)

References 19 publications

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“…In the late 1980s Malatesta and Capaldi (41) showed that iodoacetamide, at a 1:1 molar ratio, reacts exclusively with cysteine 115 on subunit III. Incubation of the enzyme with eosin-5maleimide also resulted in binding at the same site (42). In the present study, the incubation of bovine cytochrome c oxidase with fluorescein maleimide (at 1:1 molar ratio) for 20 min at 25 °C resulted in selective labeling (∼90%) of subunit III as verified by SDS gel electrophoresis.…”

Section: Resultssupporting

confidence: 65%

Proton-Collecting Properties of Bovine Heart Cytochrome c Oxidase: Kinetic and Electrostatic Analysis

et al. 2001

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Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.

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Section: Resultssupporting

confidence: 65%

Proton-Collecting Properties of Bovine Heart Cytochrome c Oxidase: Kinetic and Electrostatic Analysis

et al. 2001

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“…The reaction with iodoacetamide blocks the formation of the complex with yeast cytochrome c indicating that (2115 is on the cytoplasmic side. Muller and Azzi [37] have shown that, following pretreatment with mersalyl, eosin 5-maleimide can be specifically incorporated into subunit 111 (presumably at C115); this modification should greatly facilitate the study of subunit 111 removal and reassociation.…”

Section: Chemical Modification Ojsuhunit 111mentioning

confidence: 99%

Cytochrome-c oxidase. Subunit structure and proton pumping

Brunori¹,

et al. 1987

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“…Cys-115 of subunit III is the sulfhydryl in cytochrome oxidase most reactive to hydrophilic sulfhydryl reagents, and Cys-115 can be labeled selectively under appropriate conditions (McGeer et al, 1977;Hall et al, 1988;Malatesta & Capaldi, 1982; Muller & Azzi, 1985). Yang et al (1984) have described the synthesis of an undecagold cluster compound containing a single maleimide group which can be used to label selectively cysteine sulfhydryl groups.…”

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confidence: 99%

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

Crum

Gruys²,

Frey³

1994

Biochemistry

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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)

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Selective labeling of beef heart cytochrome oxidase subunit III with eosin‐5‐maleimide

Cited by 13 publications

References 19 publications

Proton-Collecting Properties of Bovine Heart Cytochrome c Oxidase: Kinetic and Electrostatic Analysis

Proton-Collecting Properties of Bovine Heart Cytochrome c Oxidase: Kinetic and Electrostatic Analysis

Cytochrome-c oxidase. Subunit structure and proton pumping

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

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