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|Title:||The Purification and Characterization of The Cytochrome D Containing Terminal Oxidase of Escherichia Coli (Bioenergetics, Membranes, Ultracentrifugation)|
|Author(s):||Miller, Michael Joseph|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The cytochrome d-containing terminal oxidase of Escherichia coli has been purified to at least 90% of homogeneity, judging from the Coomassie blue staining of SDS-polyacrylamide gels. These gels showed that the cytochrome contained two types of subunits with molecular weights of 57,000 Daltons and 43,000 Daltons from the analysis of a Ferguson plot, or 53,000 Daltons and 28,000 Daltons from the analysis of 12.5% gels. Reduced minus oxidized spectra of the cytochrome showed that cytochromes a(,1), b(,558) and d are present. Protoheme IX and heme d were the only prosthetic groups found in the complex, and iron was the only metal. This suggests that cythochrome a(,1) contains protoheme IX as a prosthetic group. Heme d is probably the site of oxygen binding, since both carbon monoxide and oxygen perturb its spectrum.
The molecular weight of the purified complex was measured by sedimentation equilibrium to be 309,000 Daltons. It contained lipopolysaccharide and Triton X-100 as well as protein. The amount of protein in the complex was 130,000 (+OR-) 20,000 Daltons. When the results of cross-linking experiments are taken into account, a subunit stoichiometry of (alpha)(beta)(,2) is the only possibility. This stoichiometry fits the data whether the subunit molecular weights from a Ferguson plot or a 12.5% gel are used. The amount of protein in the complex was also used to calculate the number of prosthetic groups it contained. There are 2 or 3 protohemes IX and 4 or 5 iron atoms per complex. Analysis of the heme d-to-protein ratio by other workers indicates that there are 2 hemes d per complex.
The cytochrome was found to be 70% as active in the purified form as it was in the E. coli membrane when ubiquinol-1 was used as a substrate. Reconstitution of the cytochrome into artificial liposomes along with ubiquinol-8 and pyruvate oxidase showed that ubiquinol-8 is probably the natural substrate for the oxidase. This experiment also showed that the respiratory chain of E. coli is probably very simple, consisting of a dehydrogenase complex of one or two proteins that donates electrons to ubiquinone-8. The ubiquinone-8 is then oxidized by the terminal oxidase complex, which consists of three proteins. The reconstituted system was also used to show that the major product of oxygen reduction by the cytochrome is water.
The cytochrome bd in artificial liposomes was shown to produce electrical potential and pH gradients across the liposome membrane. This showed that it was capable of translocating protons. The proton-to-electron ratio was measured to be 0.85. This can be accounted for if ubiquinol is oxidized on one side of the membrane, and oxygen is reduced on the other, and does not require the active transport of protons across the membrane.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.
|Date Available in IDEALS:||2014-12-15|