Isolation and characterization of new mutants of the cytochrome d terminal oxidase of Escherichia coli
type: CreativeWork
description: The cytochrome d terminal oxidase from Escherichia coli is a two-subunit, three-heme integral membrane cytochrome. Subunit I contains the heme center $b\sb{558}$ and Subunit II, is necessary for the binding of the $b\sb{595}$ and d heme prosthetic groups. Earlier work suggests that the N-terminus of Subunit I participates in the binding of the $b\sb{595}$ and d heme centers. Mutants in the cytochrome d terminal oxidase from Escherichia coli were isolated using hydroxylamine mutagenesis. These mutants, isolated both in the chromosome and on plasmids, were characterized using reduced minus air-oxidized difference spectroscopy, Western immunoblotting, CO difference spectroscopy, and oxidase activity assays. Plasmic-encoded mutations were mapped by marker rescue and identified by DNA sequencing. Two new classes of cytochrome d terminal oxidase mutants were identified. Under aerobic growth conditions, one class produces both subunits, and binds both the $b\sb{558}$ and $b\sb{595}$ heme centers, but varying amounts of the heme d center. The mutations conferring this phenotype are in the N-terminus of Subunit II. The conclusion is that the N-terminus of Subunit II along with the N-terminus of Subunit I forms the interface between these two subunits where the $b\sb{595}$ and d heme centers bind. The second class of mutations also produces both subunits but binds only $b\sb{558}$ and $b\sb{595}$. No heme d is bound under aerobic conditions. One of these mutations was identified and is also found in the N-terminus of Subunit I confirming its involvement in the formation of the heme-binding interface. There is another phenotype associated with the above mutants. When cells containing these mutations are grown anaerobically, the stability of the mutant enzymes improved as evidenced by increased heme binding and subunit stability. During the course of this work, a technique was developed to create chromosomal replacements in E. coli. Plasmid DNA carrying the desired allele for g…
abstract: The cytochrome d terminal oxidase from Escherichia coli is a two-subunit, three-heme integral membrane cytochrome. Subunit I contains the heme center $b\sb{558}$ and Subunit II, is necessary for the binding of the $b\sb{595}$ and d heme prosthetic groups. Earlier work suggests that the N-terminus of Subunit I participates in the binding of the $b\sb{595}$ and d heme centers. Mutants in the cytochrome d terminal oxidase from Escherichia coli were isolated using hydroxylamine mutagenesis. These mutants, isolated both in the chromosome and on plasmids, were characterized using reduced minus air-oxidized difference spectroscopy, Western immunoblotting, CO difference spectroscopy, and oxidase activity assays. Plasmic-encoded mutations were mapped by marker rescue and identified by DNA sequencing. Two new classes of cytochrome d terminal oxidase mutants were identified. Under aerobic growth conditions, one class produces both subunits, and binds both the $b\sb{558}$ and $b\sb{595}$ heme centers, but varying amounts of the heme d center. The mutations conferring this phenotype are in the N-terminus of Subunit II. The conclusion is that the N-terminus of Subunit II along with the N-terminus of Subunit I forms the interface between these two subunits where the $b\sb{595}$ and d heme centers bind. The second class of mutations also produces both subunits but binds only $b\sb{558}$ and $b\sb{595}$. No heme d is bound under aerobic conditions. One of these mutations was identified and is also found in the N-terminus of Subunit I confirming its involvement in the formation of the heme-binding interface. There is another phenotype associated with the above mutants. When cells containing these mutations are grown anaerobically, the stability of the mutant enzymes improved as evidenced by increased heme binding and subunit stability. During the course of this work, a technique was developed to create chromosomal replacements in E. coli. Plasmid DNA carrying the desired allele for g…
author: Oden, Kristine Leigh
language: eng
keywords: Biology, Molecular, Biology, Genetics, Chemistry, Biochemistry
date published: 2011-05-07T13:39:43Z
date modified: 2026-07-22T22:25:19Z
provider: University of Illinois - Urbana-Champaign
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Illinois at Urbana-Champaign

PropertyValue
property ID: Handle
value: http://hdl.handle.net/2142/22434