Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity

Jie Zhang, Petra Hellwig, Jeffrey P. Osborne, Robert B. Gennis

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihyclromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O2. In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and H391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N′,N′-tetramethyl-p- phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center wliere O2 is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b558, heme b595, and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg 391 is close to Met393, one of the axial ligands to heme b558, it is to be expected that the R391A mutation might destabilize the reduced form of heme b558. The fact that the midpoint potentials of heme d and heme b595 are also significantly lowered in the M391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.

Original languageEnglish (US)
Pages (from-to)53980-53987
Number of pages8
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004
Externally publishedYes

Fingerprint

Cytochromes
Heme
Escherichia coli
Arginine
Oxidoreductases
Mutation
Oxygen
Mutagenesis
Proton-Motive Force
Reducing Agents
Pathogens
Enzymes
Site-Directed Mutagenesis
Titration
duroquinol oxidase
Protons
Bacteria
Respiration
Ligands
Membranes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity. / Zhang, Jie; Hellwig, Petra; Osborne, Jeffrey P.; Gennis, Robert B.

In: Journal of Biological Chemistry, Vol. 279, No. 52, 24.12.2004, p. 53980-53987.

Research output: Contribution to journalArticle

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title = "Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity",
abstract = "The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihyclromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O2. In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and H391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N′,N′-tetramethyl-p- phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center wliere O2 is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b558, heme b595, and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg 391 is close to Met393, one of the axial ligands to heme b558, it is to be expected that the R391A mutation might destabilize the reduced form of heme b558. The fact that the midpoint potentials of heme d and heme b595 are also significantly lowered in the M391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.",
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T1 - Arginine 391 in subunit I of the cytochrome bd quinol oxidase from Escherichia coli stabilizes the reduced form of the hemes and is essential for quinol oxidase activity

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N2 - The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihyclromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O2. In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and H391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N′,N′-tetramethyl-p- phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center wliere O2 is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b558, heme b595, and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg 391 is close to Met393, one of the axial ligands to heme b558, it is to be expected that the R391A mutation might destabilize the reduced form of heme b558. The fact that the midpoint potentials of heme d and heme b595 are also significantly lowered in the M391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.

AB - The cytochrome bd quinol oxidase is one of two respiratory oxidases in Escherichia coli. It oxidizes dihydroubiquinol or dihyclromenaquinol while reducing dioxygen to water. The bd-type oxidases have only been found in prokaryotes and have been implicated in the survival of some bacteria, including pathogens, under conditions of low aeration. With a high affinity for dioxygen, cytochrome bd not only couples respiration to the generation of a proton motive force but also scavenges O2. In the current work, the role of a highly conserved arginine residue is explored by site-directed mutagenesis. Four mutations were made: R391A, R391K, R391M, and H391Q. All of the mutations except R391K result in enzyme lacking ubiquinol oxidase activity. Oxidase activity using the artificial reductant N,N,N′,N′-tetramethyl-p- phenylenediamine in place of ubiquinol was, however, unimpaired by the mutations, indicating that the catalytic center wliere O2 is reduced is intact. UV-visible spectra of each of the mutant oxidases show no perturbations to any of the three heme components (heme b558, heme b595, and heme d). However, spectroelectrochemical titrations of the R391A mutant reveal that the midpoint potentials of all of the heme components are substantially lower compared with the wild type enzyme. Since Arg 391 is close to Met393, one of the axial ligands to heme b558, it is to be expected that the R391A mutation might destabilize the reduced form of heme b558. The fact that the midpoint potentials of heme d and heme b595 are also significantly lowered in the M391A mutant is consistent with these hemes being physically close together on the periplasmic side of the membrane.

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